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Sample records for activating downstream signaling

  1. Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

    PubMed

    Balzano, Deborah; Fawal, Mohamad-Ali; Velázquez, Jose V; Santiveri, Clara M; Yang, Joshua; Pastor, Joaquín; Campos-Olivas, Ramón; Djouder, Nabil; Lietha, Daniel

    2015-10-01

    Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects. PMID:26286748

  2. Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation.

    PubMed

    Wang, Chunhe; Mooney, Jeffery L; Meza-Romero, Roberto; Chou, Yuan K; Huan, Jianya; Vandenbark, Arthur A; Offner, Halina; Burrows, Gregory G

    2003-08-15

    Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells. PMID:12902496

  3. Fn14, a Downstream Target of the TGF-β Signaling Pathway, Regulates Fibroblast Activation

    PubMed Central

    Yang, Min; Lai, Wen; Ye, Litong; Chen, Jing; Hou, Xinghua; Ding, Hong; Zhang, Wenwei; Wu, Yueheng; Liu, Xiaoying; Huang, Shufang; Yu, Xiyong; Xiao, Dingzhang

    2015-01-01

    Fibrosis, the hallmark of human injuries and diseases such as serious burns, is characterized by excessive collagen synthesis and myofibroblast accumulation. Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, has been implicated in fibrosis in animals. In addition to TGF-β, fibroblast growth factor-inducible molecule 14 (Fn14) has been reported to play an important role in fibrotic diseases, such as cardiac fibrosis. However, the function and detailed regulatory mechanism of Fn14 in fibrosis are unclear. Here, we investigated the effect of Fn14 on the activation of human dermal fibroblasts. In normal dermal fibroblasts, TGF-β signaling increased collagen production and Fn14 expression. Furthermore, Fn14 siRNA blocked extracellular matrix gene expression; even when TGF-β signaling was activated by TGF-β1, fibroblast activation remained blocked in the presence of Fn14 siRNA. Overexpressing Fn14 increased extracellular matrix gene expression. In determining the molecular regulatory mechanism, we discovered that SMAD4, an important TGF-β signaling co-mediator, bound to the Fn14 promoter and activated Fn14 transcription. Taken together, these results indicate that the TGF-β signaling pathway activates Fn14 expression through the transcription factor SMAD4 and that activated Fn14 expression increases extracellular matrix synthesis and fibroblast activation. Therefore, Fn14 may represent a promising approach to preventing the excessive accumulation of collagen or ECM in skin fibrosis. PMID:26625141

  4. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

    PubMed Central

    Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

    2013-01-01

    Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

  5. Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes.

    PubMed

    van Unen, Jakobus; Rashidfarrokhi, Ali; Hoogendoorn, Eelco; Postma, Marten; Gadella, Theodorus W J; Goedhart, Joachim

    2016-09-01

    Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves. PMID:27358232

  6. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  7. Suramin blocks interaction between human FGF1 and FGFR2 D2 domain and reduces downstream signaling activity.

    PubMed

    Wu, Zong-Sian; Liu, Che Fu; Fu, Brian; Chou, Ruey-Hwang; Yu, Chin

    2016-09-01

    The extracellular portion of the human fibroblast growth factor receptor2 D2 domain (FGFR2 D2) interacts with human fibroblast growth factor 1 (hFGF1) to activate a downstream signaling cascade that ultimately affects mitosis and differentiation. Suramin is an antiparasiticdrug and a potent inhibitor of FGF-induced angiogenesis. Suramin has been shown to bind to hFGF1, and might block the interaction between hFGF1 and FGFR2 D2. Here, we titrated hFGF1 with FGFR2 D2 and suramin to elucidate their interactions using the detection of NMR. The docking results of both hFGF1-FGFR2 D2 domain and hFGF1-suramin complex were superimposed. The results indicate that suramin blocks the interaction between hFGF1 and FGFR2 D2. We used the PyMOL software to show the hydrophobic interaction of hFGF1-suramin. In addition, we used a Water-soluble Tetrazolium salts assay (WST1) to assess hFGF1 bioactivity. The results will be useful for the development of new antimitogenic activity drugs. PMID:27387234

  8. Pyrvinium attenuates Hedgehog signaling downstream of smoothened.

    PubMed

    Li, Bin; Fei, Dennis Liang; Flaveny, Colin A; Dahmane, Nadia; Baubet, Valérie; Wang, Zhiqiang; Bai, Feng; Pei, Xin-Hai; Rodriguez-Blanco, Jezabel; Hang, Brian; Orton, Darren; Han, Lu; Wang, Baolin; Capobianco, Anthony J; Lee, Ethan; Robbins, David J

    2014-09-01

    The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds, vismodegib, is now FDA-approved for patients with advanced basal cell carcinoma, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50, 10 nmol/L) casein kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib -resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator suppressor of fused. We go on to demonstrate the utility of this small molecule in vivo, against the HH-dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers. PMID:24994715

  9. Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

    PubMed

    Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

    2014-09-26

    We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. PMID:25148941

  10. Increased Cortical Synaptic Activation of TrkB and Downstream Signaling Markers in a Mouse Model of Down Syndrome

    PubMed Central

    Nosheny, RL; Belichenko, PV; Busse, BL; Weissmiller, AM; Dang, V; Das, D; Fahimi, A; Salehi, A; Smith, SJ; Mobley, WC

    2015-01-01

    Down Syndrome (DS), trisomy 21, is characterized by synaptic abnormalities and cognitive deficits throughout the lifespan and with development of Alzheimer’s disease (AD) neuropathology and progressive cognitive decline in adults. Synaptic abnormalities are also present in the Ts65Dn mouse model of DS, but which synapses are affected and the mechanisms underlying synaptic dysfunction are unknown. Here we show marked increases in the levels and activation status of TrkB and associated signaling proteins in cortical synapses in Ts65Dn mice. Proteomic analysis at the single synapse level of resolution using array tomography (AT) uncovered increased colocalization of activated TrkB with signaling endosome related proteins, and demonstrated increased TrkB signaling. The extent of increases in TrkB signaling differed in each of the cortical layers examined and with respect to the type of synapse, with the most marked increases seen in inhibitory synapses. These findings are evidence of markedly abnormal TrkB-mediated signaling in synapses. They raise the possibility that dysregulated TrkB signaling contributes to synaptic dysfunction and cognitive deficits in DS. PMID:25753471

  11. Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

    PubMed Central

    Proksch, Peter; Abel, Josef; Dvorak, Zdenek; Haarmann-Stemmann, Thomas

    2013-01-01

    Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1), which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE)-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones. PMID:24069365

  12. GPR30 Promotes Prostate Stromal Cell Activation via Suppression of ERα Expression and Its Downstream Signaling Pathway.

    PubMed

    Jia, Bona; Gao, Yu; Li, Mingming; Shi, Jiandang; Peng, Yanfei; Du, Xiaoling; Klocker, Helmut; Sampson, Natalie; Shen, Yongmei; Liu, Mengyang; Zhang, Ju

    2016-08-01

    Cancer-associated fibroblasts (CAFs) play a vital role in malignant transformation and progression of prostate cancer (PCa), and accumulating evidence suggests an enhancing effect of estrogens on PCa. The present study aimed to investigate the possible origin of prostate CAFs and the effects of estrogen receptors, G protein-coupled receptor 30 (GPR30) and estrogen receptor (ER)-α, on stromal cell activation. High expression of fibroblast activation protein (FAP), CD44, and nonmuscle myosin heavy chain B (SMemb) accompanied by low expression of smooth muscle differentiation markers was found in the stromal cells of PCa tissues and in cultured human prostate CAFs. Additionally, SMemb expression, which is coupled to cell phenotype switching and proliferation, was coexpressed with FAP, a marker of activated stromal cells, and with the stem cell marker CD44 in the stromal cells of PCa tissue. Prostate CAFs showed high GPR30 and low ERα expression. Moreover, GPR30 was coexpressed with FAP, CD44, and SMemb. Furthermore, the study demonstrated that the overexpression of GPR30 or the knockdown of ERα in prostate stromal cells induced the up-regulation of FAP, CD44, Smemb, and the down-regulation of smooth muscle markers. The conditioned medium from these cells promoted the proliferation and migration of LNCaP and PC3 PCa cells. GPR30 knockdown or ERα overexpression showed opposite effects. Finally, we present a novel mechanism whereby GPR30 limits ERα expression via inhibition of the cAMP/protein kinase A signaling pathway. These results suggest that stem-like cells within the stroma are a possible source of prostate CAFs and that the negative regulation of ERα expression by GPR30 is centrally involved in prostate stromal cell activation. PMID:27163843

  13. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  14. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    PubMed

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  15. Serine-71 phosphorylation of Rac1 modulates downstream signaling.

    PubMed

    Schwarz, Janett; Proff, Julia; Hävemeier, Anika; Ladwein, Markus; Rottner, Klemens; Barlag, Britta; Pich, Andreas; Tatge, Helma; Just, Ingo; Gerhard, Ralf

    2012-01-01

    The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways. PMID:22970203

  16. Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    PubMed Central

    Akhtar, Saghir; Yousif, Mariam H. M.; Dhaunsi, Gursev S.; Sarkhouh, Fatma; Chandrasekhar, Bindu; Attur, Sreeja; Benter, Ibrahim F.

    2013-01-01

    Diabetes mellitus leads to vascular complications but the underlying signalling mechanisms are not fully understood. Here, we examined the role of ErbB2 (HER2/Neu), a transmembrane receptor tyrosine kinase of the ErbB/EGFR (epidermal growth factor receptor) family, in mediating diabetes-induced vascular dysfunction in an experimental model of type 1 diabetes. Chronic treatment of streptozotocin-induced diabetic rats (1 mg/kg/alt diem) or acute, ex-vivo (10−6, 10−5 M) administration of AG825, a specific inhibitor of ErbB2, significantly corrected the diabetes-induced hyper-reactivity of the perfused mesenteric vascular bed (MVB) to the vasoconstrictor, norephinephrine (NE) and the attenuated responsiveness to the vasodilator, carbachol. Diabetes led to enhanced phosphorylation of ErbB2 at multiple tyrosine (Y) residues (Y1221/1222, Y1248 and Y877) in the MVB that could be attenuated by chronic AG825 treatment. Diabetes- or high glucose-mediated upregulation of ErbB2 phosphorylation was coupled with activation of Rho kinases (ROCKs) and ERK1/2 in MVB and in cultured vascular smooth muscle cells (VSMC) that were attenuated upon treatment with either chronic or acute AG825 or with anti-ErbB2 siRNA. ErbB2 likley heterodimerizes with EGFR, as evidenced by increased co-association in diabetic MVB, and further supported by our finding that ERK1/2 and ROCKs are common downstream effectors since their activation could also be blocked by AG1478. Our results show for the first time that ErbB2 is an upstream effector of ROCKs and ERK1/2 in mediating diabetes-induced vascular dysfunction. Thus, potential strategies aimed at modifying actions of signal transduction pathways involving ErbB2 pathway may prove to be beneficial in treatment of diabetes-induced vascular complications. PMID:23826343

  17. Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive downstream signaling networks

    PubMed Central

    Chakraborty, Sharmistha; Li, Li; Puliyappadamba, VineshkumarThidil; Guo, Gao; Hatanpaa, Kimmo J.; Mickey, Bruce; Souza, Rhonda F.; Vo, Peggy; Herz, Joachim; Chen, Mei-Ru; Boothman, David A.; Pandita, Tej K.; Wang, David H.; Sen, Ganes C.; Habib, Amyn A.

    2014-01-01

    EGFR overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling. Constitutive or non-canonical EGFR signaling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1, and TRAIL. Ligand-mediated activation of EGFR switches off IRF3 dependent transcription, activates canonical ERK and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of EGF dissociates TBK1, IRF3, and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signaling in glioblastoma. PMID:25503978

  18. Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

    PubMed Central

    Hashimoto, Ayako

    2015-01-01

    Background Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells. Methods and Results Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis. Conclusions The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis. PMID:26181635

  19. Turbulence decay downstream of an active grid

    NASA Astrophysics Data System (ADS)

    Bewley, Gregory; Bodenschatz, Eberhard

    2015-11-01

    A grid in a wind tunnel stirs up turbulence that has a certain large-scale structure. The moving parts in a so-called ``active grid'' can be programmed to produce different structures. We use a special active grid in which each of 129 paddles on the grid has its own position-controlled servomotor that can move independently of the others. We observe among other things that the anisotropy in the amplitude of the velocity fluctuations and in the correlation lengths can be set and varied with an algorithm that oscillates the paddles in a specified way. The variation in the anisotropies that we observe can be explained by our earlier analysis of anisotropic ``soccer ball'' turbulence (Bewley, Chang and Bodenschatz 2012, Phys. Fluids). We define the influence of this variation in structure on the downstream evolution of the turbulence. with Eberhard Bodenschatz and others.

  20. DNA G-quadruplex formation in response to remote downstream transcription activity: long-range sensing and signal transducing in DNA double helix.

    PubMed

    Zhang, Chao; Liu, Hong-He; Zheng, Ke-Wei; Hao, Yu-Hua; Tan, Zheng

    2013-08-01

    G-quadruplexes, four-stranded structures formed by Guanine-rich nucleic acids, are implicated in many physiological and pathological processes. G-quadruplex-forming sequences are abundant in genomic DNA, and G-quadruplexes have recently been shown to exist in the genome of mammalian cells. However, how G-quadruplexes are formed in the genomes remains largely unclear. Here, we show that G-quadruplex formation can be remotely induced by downstream transcription events that are thousands of base pairs away. The induced G-quadruplexes alter protein recognition and cause transcription termination at the local region. These results suggest that a G-quadruplex-forming sequence can serve as a sensor or receiver to sense remote DNA tracking activity in response to the propagation of mechanical torsion in a DNA double helix. We propose that the G-quadruplex formation may provide a mean for long-range sensing and communication between distal genomic locations to coordinate regulatory transactions in genomic DNA. PMID:23716646

  1. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling

    PubMed Central

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C.; Perino, Alessia; Longo, Dario L.; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D.; Backer, Jonathan M.; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  2. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling.

    PubMed

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C; Perino, Alessia; Longo, Dario L; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D; Backer, Jonathan M; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  3. Myricetin suppresses p21-activated kinase 1 in human breast cancer MCF-7 cells through downstream signaling of the β-catenin pathway.

    PubMed

    Jiao, De; Zhang, Xue Dong

    2016-07-01

    As a main active compound in the bark of waxberry (Myrica rubra), myricetin is a macrocyclic diarylheptanoid, and can trigger the apoptosis of HeLa and PC3 cells. The aim of the present study was to elucidate the anticancer effect of myricetin on human breast cancer MCF-7 cells and to explore the possible mechanisms of action. MCF-7 cells were treated with different concentrations of myricetin (0-80 µM) for 12, 24 and 48 h. In the present study, we found that myricetin suppressed the cell viability of the MCF-7 cells at least partly through the induction of apoptosis as determined by MTT assay and flow cytometry. Western blot analysis revealed that myricetin effectively suppressed the protein expression of p21-activated kinase 1 (PAK1), MEK and phosphorylated extracellular mitogen-activated protein kinase (ERK1/2). In addition, treatment of myricetin activated glycogen synthase kinase-3β (GSK3β) and Bax protein expression, and inhibited β-catenin/cyclin D1/proliferating cell nuclear antigen (PCNA)/survivin and promoted caspase-3 activity in the MCF-7 cells. These results demonstrated that myricetin suppressed the cell viability of human breast cancer MCF-7 cells through PAK1/MEK/ERK/GSK3β/β-catenin/cyclin D1/PCNA/survivin/Bax-caspase-3 signaling. PMID:27122002

  4. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    SciTech Connect

    Kim, Kyung-Chang; Kim, Hyeon Guk; Roh, Tae-Young; Park, Jihwan; Jung, Kyung-Min; Lee, Joo-Shil; Choi, Sang-Yun; Kim, Sung Soon; Choi, Byeong-Sun

    2011-01-14

    Research highlights: {yields} CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. {yields} CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. {yields} HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. {yields} H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. {yields} HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56{sup Lck}, ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56{sup Lck}, ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new

  5. EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells

    SciTech Connect

    Rodriguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie G.; Lauer, Fredine T.; Burchiel, Scott W.

    2009-03-15

    Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-{gamma}1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 {mu}M), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-{gamma}1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-{gamma}1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-{gamma}1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

  6. Oxidative stress and its downstream signaling in aging eyes

    PubMed Central

    Pinazo-Durán, María Dolores; Gallego-Pinazo, Roberto; García-Medina, Jose Javier; Zanón-Moreno, Vicente; Nucci, Carlo; Dolz-Marco, Rosa; Martínez-Castillo, Sebastián; Galbis-Estrada, Carmen; Marco-Ramírez, Carla; López-Gálvez, Maria Isabel; Galarreta, David J; Díaz-Llópis, Manuel

    2014-01-01

    Background Oxidative stress (OS) and its biomarkers are the biochemical end point of the imbalance between reactive oxygen species (ROS) production and the ability of the antioxidant (AOX) biological systems to fight against oxidative injury. Objective We reviewed the role of OS and its downstream signaling in aging eyes. Methods A search of the literature and current knowledge on the physiological and pathological mechanisms of OS were revisited in relation to the eyes and the aging process. Most prevalent ocular diseases have been analyzed herein in relation to OS and nutraceutic supplements, such as dry-eye disorders, glaucoma, age-related macular degeneration, and diabetic retinopathy. Results Clinical, biochemical, and molecular data from anterior and posterior eye segment diseases point to OS as the common pathogenic mechanism in the majority of these ocular disorders, many of which are pathologies causing visual impairment, blindness, and subsequent loss of life quality. Studies with nutraceutic supplements in aging eye-related pathologies have also been reviewed. Conclusion OS, nutritional status, and nutraceutic supplements have to be considered within the standards of care of older ophthalmologic patients. OS biomarkers and surrogate end points may help in managing the aging population with ocular diseases. PMID:24748782

  7. OxLDL-Dependent Activation of Arginase II Is Dependent on the LOX-1 Receptor and Downstream RhoA Signaling

    PubMed Central

    Ryoo, Sungwoo; Bhunia, Anil; Chang, Fumin; Shoukas, Artin; Berkowitz, Dan E; Romer, Lewis H

    2010-01-01

    Aims Arginase II regulates NOS activity by competing for the substrate L-arginine. Oxidized LDL (OxLDL) is a proatherogenic molecule that activates arginase II. We tested the hypotheses that OxLDL-dependent arginase II activation occurs through a specific receptor, and via a Rho GTPase effector mechanism that is inhibited by statins. Methods and Results Arginase II activation by OxLDL was attenuated following preincubation with the LOX-1 receptor-blocking antibody JTX92. This also prevented the dissociation of arginase II from microtubules. LOX-1−/− mice failed to exhibit the increased arginase II activity seen in WT mice fed a high cholesterol diet. Furthermore, endothelium from LOX−/− mice failed to demonstrate the diet-dependent reduction in NO and increase in ROS that were observed in WT mice. OxLDL induced Rho translocation to the membrane and Rho activation, and these effects were inhibited by pretreatment with JTX92 or statins. Transfection with siRNA for RhoA, or inhibition of ROCK both decreased OxLDL-stimulated arginase II activation. Preincubation with simvastatin or lovastatin blocked OxLDL-induced dissociation of arginase II from microtubules and prevented microtubule depolymerization. Conclusions This study provides a new focus for preventive therapy for atherosclerotic disease by delineating a clearer path from OxLDL through the endothelial cell LOX-1 receptor, RhoA, and ROCK, to the activation of arginase II, downregulation of NO, and vascular dysfunction. PMID:21130456

  8. Lipoteichoic acid-deficient Lactobacillus acidophilus regulates downstream signals.

    PubMed

    Saber, Rana; Zadeh, Mojgan; Pakanati, Krishna C; Bere, Praveen; Klaenhammer, Todd; Mohamadzadeh, Mansour

    2011-03-01

    The trillions of microbes residing within the intestine induce critical signals that either regulate or stimulate host immunity via their bacterial products. To better understand the immune regulation elicited by lipoteichoic acid (LTA)-deficient Lactobacillus acidophilus NCFM in steady state and induced inflammation, we deleted phosphoglycerol transferase gene, which synthesizes LTA in L. acidophilus NCFM. In vitro and in vivo experiments were conducted in order to compare the immune regulatory properties of the L. acidophilus strain deficient in LTA (NCK2025) with its wild-type parent (NCK56) in C57BL/6, C57BL/6 recombination-activation gene 1-deficient (Rag1 (-/-)) and C57BL/6 Rag1(-/-)IL-10(-/-) mice. We demonstrate that NCK2025 significantly activates the phosphorylation of Erk1/2 but downregulates the phosphorylation of Akt1, cytosolic group IV PLA2 and p38 in mouse dendritic cells. Similarly, mice treated orally with NCK2025 exhibit decreased phosphorylation of inflammatory signals (Akt1, cytosolic group IV PLA2 or P38) but upregulate Erk1/2-phosphorylation in colonic epithelial cells in comparison with mice treated with NCK56. In addition, regulation of pathogenic CD4+ T cell induced colitis by NCK2025 was observed in Rag1 (-/-) but not Rag1(-/-)IL-10 (-/-) mice suggests a critical role of IL-10 that may be tightly regulated by Erk1/2 signaling. These data highlight the immunosuppressive properties of NCK2025 to deliver regulatory signals in innate cells, which results in the mitigation of T-cell-induced colitis in vivo. PMID:21395377

  9. EVALUATING THE EFFECT OF UPSTREAM WATERSHED ACTIVITIES TO DOWNSTREAM STREAMFLOW

    EPA Science Inventory

    Linking the impacts of upstream activities such as urban development to changes in downstream streamflow is critical to achieving a balance between economic development and environmental protection as a basis for sustainable watershed development. This paper presents a modeling a...

  10. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

    PubMed Central

    Ray, Poulomi; Chapman, Susan C.

    2015-01-01

    Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. PMID:26237312

  11. Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

    NASA Astrophysics Data System (ADS)

    Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

    2014-03-01

    During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

  12. Sbds is required for Rac2-mediated monocyte migration and signaling downstream of RANK during osteoclastogenesis.

    PubMed

    Leung, Roland; Cuddy, Karl; Wang, Yongqiang; Rommens, Johanna; Glogauer, Michael

    2011-02-10

    Shwachman-Diamond syndrome (SDS) results from mutations in the SBDS gene, characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. Neutropenia and neutrophil dysfunction are hallmark features of SDS; however, causes for the bone defects are unknown. Dysfunction of bone-resorbing osteoclasts, formed by the fusion of monocytic progenitors derived from the same granulocytic precursors as neutrophils, could be responsible. We report that Sbds is required for in vitro and in vivo osteoclastogenesis (OCG). Sbds-null murine monocytes formed osteoclasts of reduced number and size because of impaired migration and fusion required for OCG. Phenotypically, Sbds-null mice exhibited low-turnover osteoporosis consistent with findings in SDS patients. Western blotting of Rho GTPases that control actin dynamics and migration showed a 5-fold decrease in Rac2, whereas Rac1, Cdc42, and RhoA were unchanged or only mildly reduced. Although migration was rescued on Rac2 supplementation, OCG was not. This was attributed to impaired signaling downstream of receptor activator of nuclear factor-κB (RANK) and reduced expression of the RANK-ligand-dependent fusion receptor DC-STAMP. We conclude that Sbds is required for OCG by regulating monocyte migration via Rac2 and osteoclast differentiation signaling downstream of RANK. Impaired osteoclast formation could disrupt bone homeostasis, resulting in skeletal abnormalities seen in SDS patients. PMID:21084708

  13. Transduction of Functionally Contrasting Signals by Two Mycobacterial PPE Proteins Downstream of TLR2 Receptors.

    PubMed

    Udgata, Atul; Qureshi, Rahila; Mukhopadhyay, Sangita

    2016-09-01

    As pathogen-associated molecular pattern sensors, the TLRs can detect diverse ligands to elicit either proinflammatory or anti-inflammatory responses, but the mechanism that dictates such contrasting immune responses is not well understood. In this work, we demonstrate that proline-proline-glutamic acid (PPE)17 protein of Mycobacterium tuberculosis induces TLR1/2 heterodimerization to elicit proinflammatory-type response, whereas PPE18-induced homodimerization of TLR2 triggers anti-inflammatory type responses. Ligation of TLR1/2 caused an increased recruitment of IL-1R-associated kinase (IRAK)1, MyD88, and protein kinase C (PKC)ε to the downstream TLR-signaling complex that translocated PKCε into the nucleus in an IRAK1-dependent manner. PKCε-mediated phosphorylation allowed the nuclear IRAK3 to be exported to the cytoplasm, leading to increased activation of ERK1/2, stabilization of MAPK phosphatase 1 (MKP-1), and induction of TNF-α with concomitant downregulation of p38MAPK. Silencing of TLR1 inhibited PPE17-triggered cytoplasmic export of IRAK3 as well as TNF-α induction, suggesting an important role of TLR1/2 heterodimer in regulating proinflammatory responses via the IRAK3-signaling pathway. In contrast, PPE18-mediated homodimerization of TLR2 caused poorer cytoplasmic export of nuclear IRAK3 and MKP-1 stabilization, resulting in increased p38MAPK activation. Our study hints to a novel mechanism that implicates PKCε-IRAK3-MKP-1 signaling in the regulation of MAPK activity and inflammatory cascades downstream of TLR2 in tuberculosis. PMID:27481848

  14. Role of mTOR Downstream Effector Signaling Molecules in Francisella Tularensis Internalization by Murine Macrophages

    PubMed Central

    Edwards, Michael W.; Aultman, James A.; Harber, Gregory; Bhatt, Jay M.; Sztul, Elizabeth; Xu, Qingan; Zhang, Ping; Michalek, Suzanne M.; Katz, Jannet

    2013-01-01

    Francisella tularensis is an infectious, gram-negative, intracellular microorganism, and the cause of tularemia. Invasion of host cells by intracellular pathogens like Francisella is initiated by their interaction with different host cell membrane receptors and the rapid phosphorylation of different downstream signaling molecules. PI3K and Syk have been shown to be involved in F. tularensis host cell entry, and both of these signaling molecules are associated with the master regulator serine/threonine kinase mTOR; yet the involvement of mTOR in F. tularensis invasion of host cells has not been assessed. Here, we report that infection of macrophages with F. tularensis triggers the phosphorylation of mTOR downstream effector molecules, and that signaling via TLR2 is necessary for these events. Inhibition of mTOR or of PI3K, ERK, or p38, but not Akt signaling, downregulates the levels of phosphorylation of mTOR downstream targets, and significantly reduces the number of F. tularensis cells invading macrophages. Moreover, while phosphorylation of mTOR downstream effectors occurs via the PI3K pathway, it also involves PLCγ1 and Ca2+ signaling. Furthermore, abrogation of PLC or Ca2+ signaling revealed their important role in the ability of F. tularensis to invade host cells. Together, these findings suggest that F. tularensis invasion of primary macrophages utilize a myriad of host signaling pathways to ensure effective cell entry. PMID:24312679

  15. Maternal diabetes promotes mTORC1 downstream signalling in rabbit preimplantation embryos.

    PubMed

    Gürke, Jacqueline; Schindler, Maria; Pendzialek, S Mareike; Thieme, René; Grybel, Katarzyna J; Heller, Regine; Spengler, Katrin; Fleming, Tom P; Fischer, Bernd; Navarrete Santos, Anne

    2016-05-01

    The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets in in vitro cultured preimplantation rabbit blastocysts and in vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1; CCND1), translation initiation (eukaryotic translation initiation factor 4E; EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy. PMID:26836250

  16. Targeting the cis-dimerization of LINGO-1 with low MW compounds affects its downstream signalling

    PubMed Central

    Cobret, L; De Tauzia, M L; Ferent, J; Traiffort, E; Hénaoui, I; Godin, F; Kellenberger, E; Rognan, D; Pantel, J; Bénédetti, H; Morisset-Lopez, S

    2015-01-01

    Background and Purpose The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. Experimental Approach LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein–protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. Key Results LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Conclusions and Implications Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery. PMID:25257685

  17. Re-modulated technology of WDM-PON employing different DQPSK downstream signals

    NASA Astrophysics Data System (ADS)

    Gao, Chao; Xin, Xiang-jun; Yu, Chong-xiu

    2012-11-01

    This paper proposes a kind of modulation architecture for wavelength-division-multiplexing passive optical network (WDMPON) employing optical differential quadrature phase shift keying (DQPSK) downstream signals and two different modulation formats of re-modulated upstream signals. At the optical line terminal (OLT), 10 Gbit/s signal is modulated with DQPSK. At the optical network unit (ONU), part of the downstream signal is re-modulated with on-off keying (OOK) or inverse-return-to-zero (IRZ). Simulation results show the impact on the system employing NRZ, RZ and carrier-suppressed return-to-zero (CSRZ). The analyses also reflect that the architecture can restrain chromatic dispersion and channel crosstalk, which makes it the best architecture of access network in the future.

  18. Misoprostol Reverse Hippocampal Neuron Cyclooxygenase-2 Downstream Signaling Imbalance in Aluminum-Overload Rats.

    PubMed

    Guo, Yuanxin; Lei, Wenjuan; Wang, Jianfeng; Hu, Xinyue; Wei, Yuling; Ji, Chaonan; Yang, Junqing

    2016-01-01

    Although COX-2 inhibition in animal models of neurodegenerative diseases has shown neuroprotection, recent studies have revealed some serious side effects (ulcers, bleeding, fatal cerebrovascular diseases etc.) and the limited benefits of COX-2 inhibitors. A more focused approach is necessary to explore the therapeutic effect of the COX downstream signaling pathway in neurological research. The aim of this study was to explore the alterations of the PGES-PGE2-EP signal pathway and the effect of misoprostol on neurodegeneration by chronic aluminum-overload in rats. Adult rats were treated by intragastric administration of aluminum gluconate. The PGE2 content and expression of PGES and EPs in the hippocampi of rats were detected using ELISA, q-PCR and Western blot analysis, respectively. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the rat hippocampi were also detected. The misoprostol treatment dose-dependently improved spatial learning and memory function as well as healing after hippocampal neuron damage induced by chronic aluminum-overload in rats. Meanwhile, the administration of misoprostol resulted in a decrease in the PGE2 level and down-regulation of the mPGES-1, EP2 and EP4 expression levels, while there was a dosedependent up-regulation of EP3 expression. These results suggest that misoprostol possesses a neuroprotective property, and the mechanism involves affecting the EP3 level and reducing the endogenous production of PGE2 through a negative feedback mechanism, increasing the EP3 expression level, decreasing the EP2 and EP4 expression levels, and rebuilding the mPGES-1-PGE2-EP1-4 signal pathway balance. In this way, misoprostol has a counteractive effect on oxidant stress and inflammation in the central nervous system. The PGES-PGE2-EPs signaling pathway is a potential therapeutic strategy for treating neurodegeneration in patients. PMID:27033056

  19. Misoprostol Reverse Hippocampal Neuron Cyclooxygenase-2 Downstream Signaling Imbalance in Aluminum-Overload Rats

    PubMed Central

    Guo, Yuanxin; Lei, Wenjuan; Wang, Jianfeng; Hu, Xinyue; Wei, Yuling; Ji, Chaonan; Yang, Junqing

    2016-01-01

    Although COX-2 inhibition in animal models of neurodegenerative diseases has shown neuroprotection, recent studies have revealed some serious side effects (ulcers, bleeding, fatal cerebrovascular diseases etc.) and the limited benefits of COX-2 inhibitors. A more focused approach is necessary to explore the therapeutic effect of the COX downstream signaling pathway in neurological research. The aim of this study was to explore the alterations of the PGES-PGE2-EP signal pathway and the effect of misoprostol on neurodegeneration by chronic aluminum-overload in rats. Adult rats were treated by intragastric administration of aluminum gluconate. The PGE2 content and expression of PGES and EPs in the hippocampi of rats were detected using ELISA, q-PCR and Western blot analysis, respectively. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the rat hippocampi were also detected. The misoprostol treatment dose-dependently improved spatial learning and memory function as well as healing after hippocampal neuron damage induced by chronic aluminum-overload in rats. Meanwhile, the administration of misoprostol resulted in a decrease in the PGE2 level and down-regulation of the mPGES-1, EP2 and EP4 expression levels, while there was a dose-dependent up-regulation of EP3 expression. These results suggest that misoprostol possesses a neuroprotective property, and the mechanism involves affecting the EP3 level and reducing the endogenous production of PGE2 through a negative feedback mechanism, increasing the EP3 expression level, decreasing the EP2 and EP4 expression levels, and rebuilding the mPGES-1-PGE2-EP1-4 signal pathway balance. In this way, misoprostol has a counteractive effect on oxidant stress and inflammation in the central nervous system. The PGES-PGE2-EPs signaling pathway is a potential therapeutic strategy for treating neurodegeneration in patients. PMID:27033056

  20. MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

    SciTech Connect

    Wang, Yelin; Hu, Chen; Cheng, Jun; Chen, Binquan; Ke, Qinghong; Lv, Zhen; Wu, Jian; Zhou, Yanfeng

    2014-04-18

    Highlights: • MiR-145 expression is down-regulated in HCC tissues and inversely related with IRS1 levels. • MiR-145 directly targets IRS1 in HCC cells. • Restored expression of miR-145 suppressed HCC cell proliferation and growth. • MiR-145 induced IRS1 under-expression potentially reduced downstream AKT signaling. - Abstract: Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

  1. Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants

    PubMed Central

    Walker, Robin K.; Zhao, Yichen; Berkowitz, Gerald A.

    2012-01-01

    Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca2+ elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca2+ elevation is attributable to cGMP activation of a Ca2+ channel. Some differences were identified between Pep/PEPR signaling and the Ca2+-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca2+ stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca2+. Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca2+ elevation, H2O2 generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen. PMID:23150556

  2. Electrical Motor Current Signal Analysis using a Modulation Signal Bispectrum for the Fault Diagnosis of a Gearbox Downstream

    NASA Astrophysics Data System (ADS)

    Haram, M.; Wang, T.; Gu, F.; Ball, A. D.

    2012-05-01

    Motor current signal analysis has been an effective way for many years of monitoring electrical machines themselves. However, little work has been carried out in using this technique for monitoring their downstream equipment because of difficulties in extracting small fault components in the measured current signals. This paper investigates the characteristics of electrical current signals for monitoring the faults from a downstream gearbox using a modulation signal bispectrum (MSB), including phase effects in extracting small modulating components in a noisy measurement. An analytical study is firstly performed to understand amplitude, frequency and phase characteristics of current signals due to faults. It then explores the performance of MSB analysis in detecting weak modulating components in current signals. Experimental study based on a 10kw two stage gearbox, driven by a three phase induction motor, shows that MSB peaks at different rotational frequencies can be based to quantify the severity of gear tooth breakage and the degrees of shaft misalignment. In addition, the type and location of a fault can be recognized based on the frequency at which the change of MSB peak is the highest among different frequencies.

  3. COUP-TFII acts downstream of Wnt/β-catenin signal to silence PPARγ gene expression and repress adipogenesis

    PubMed Central

    Okamura, Masashi; Kudo, Hiromi; Wakabayashi, Ken-ichi; Tanaka, Toshiya; Nonaka, Aya; Uchida, Aoi; Tsutsumi, Shuichi; Sakakibara, Iori; Naito, Makoto; Osborne, Timothy F.; Hamakubo, Takao; Ito, Sadayoshi; Aburatani, Hiroyuki; Yanagisawa, Masashi; Kodama, Tatsuhiko; Sakai, Juro

    2009-01-01

    Wnt signaling through β-catenin and TCF maintains preadipocytes in an un-differentiated proliferative state; however, the molecular pathway has not been completely defined. By integrating gene expression microarray, chromatin immunoprecipitation-chip, and cell-based experimental approaches, we show that Wnt/β-catenin signaling activates the expression of COUP-TFII which recruits the SMRT corepressor complex to the first introns located downstream from the first exons of both PPARγ1 and γ2 mRNAs. This maintains the local chromatin in a hypoacetylated state and represses PPARγ gene expression to inhibit adipogenesis. Our experiments define the COUP-TFII/SMRT complex as a previously unappreciated component of the linear pathway that directly links Wnt/β-catenin signaling to repression of PPARγ gene expression and the inhibition of adipogenesis. PMID:19307559

  4. Downstream elements of mammalian pre-mRNA polyadenylation signals: primary, secondary and higher-order structures.

    PubMed

    Zarudnaya, Margarita I; Kolomiets, Iryna M; Potyahaylo, Andriy L; Hovorun, Dmytro M

    2003-03-01

    Primary, secondary and higher-order structures of downstream elements of mammalian pre-mRNA polyadenylation signals [poly(A) signals] are re viewed. We have carried out a detailed analysis on our database of 244 human pre-mRNA poly(A) signals in order to characterize elements in their downstream regions. We suggest that the downstream region of the mammalian pre-mRNA poly(A) signal consists of various simple elements located at different distances from each other. Thus, the downstream region is not described by any precise consensus. Searching our database, we found that approximately 80% of pre-mRNAs with the AAUAAA or AUUAAA core upstream elements contain simple downstream elements, consisting of U-rich and/or 2GU/U tracts, the former occurring approximately 2-fold more often than the latter. Approximately one-third of the pre-mRNAs analyzed here contain sequences that may form G-quadruplexes. A substantial number of these sequences are located immediately downstream of the poly(A) signal. A possible role of G-rich sequences in the polyadenylation process is discussed. A model of the secondary structure of the SV40 late pre-mRNA poly(A) signal downstream region is presented. PMID:12595544

  5. Electrical motor current signal analysis using a modified bispectrum for fault diagnosis of downstream mechanical equipment

    NASA Astrophysics Data System (ADS)

    Gu, F.; Shao, Y.; Hu, N.; Naid, A.; Ball, A. D.

    2011-01-01

    This paper presents the use of the induction motor current to identify and quantify common faults within a two-stage reciprocating compressor based on bispectrum analysis. The theoretical basis is developed to understand the nonlinear characteristics of current signals when the motor undertakes a varying load under different faulty conditions. Although conventional bispectrum representation of current signal allows the inclusion of phase information and the elimination of Gaussian noise, it produces unstable results due to random phase variation of the sideband components in the current signal. A modified bispectrum based on the amplitude modulation feature of the current signal is then adopted to combine both lower sidebands and higher sidebands simultaneously and hence characterise the current signal more accurately. Based on this new bispectrum analysis a more effective diagnostic feature, namely normalised bispectral peak, is developed for fault classification. In association with the kurtosis value of the raw current signal, the bispectrum feature gives rise to reliable fault classification results. In particular, the low feature values can differentiate the belt looseness from the other fault cases and different degrees of discharge valve leakage and inter-cooler leakage can be separated easily using two linear classifiers. This work provides a novel approach to the analysis of stator current for the diagnosis of motor drive faults from downstream driving equipment.

  6. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

    PubMed Central

    Ma, Gary S.; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K.; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L.; Ghosh, Pradipta

    2015-01-01

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions. PMID:25926659

  7. Id proteins are critical downstream effectors of BMP signaling in human pulmonary arterial smooth muscle cells.

    PubMed

    Yang, Jun; Li, Xiaohui; Li, Ying; Southwood, Mark; Ye, Lingying; Long, Lu; Al-Lamki, Rafia S; Morrell, Nicholas W

    2013-08-15

    Bone morphogenetic protein type II receptor (BMPR-II) mutations are responsible for over 70% of cases of heritable pulmonary arterial hypertension (PAH). Loss of BMP signaling promotes pulmonary vascular remodeling via modulation of pulmonary artery smooth muscle cell (PASMC) proliferation. Id proteins (Id1-4) are major downstream transcriptional targets of BMP signaling. However, the impact of BMPR-II mutation on the expression of the range of Id proteins and the contribution of individual Id proteins to abnormal PASMC function remain unclear. Human PASMCs were used to determine the expression of Id proteins (Id1-4) by real-time PCR and immunoblotting. The BMP responses in control cells were compared with PASMCs harboring BMPR-II mutations and cells in which BMPR-II was knocked down by siRNA transfection. Id3 expression in pulmonary vessels was also investigated in BMPR-II mutant mice and in patients with heritable PAH. BMP4 and BMP6, but not BMP9, induced mRNA expression of Id1, Id2, and Id3. The BMP-stimulated induction of Id1 and Id3 was markedly reduced in BMPR-II mutant PASMCs and in control PASMCs following siRNA silencing of BMPR-II. Pulmonary arteries in BMPR-II mutant mice and patients with heritable PAH demonstrated reduced levels of Id3 compared with control subjects. Lentiviral overexpression of Id3 reduced cell cycle progression and inhibited proliferation of PASMCs. Lipopolysaccharide further reduced Id3 expression in mutant PASMCs. In conclusion, Id proteins, and particularly Id1 and Id3, are critical downstream effectors of BMP signaling in PASMCs. Loss of BMPR-II function reduces the induction of Id genes in PASMCs, Id1, and Id3 regulate the proliferation of PASMCs via cell cycle inhibition, an effect that may be exacerbated by inflammatory stimuli. PMID:23771884

  8. Id proteins are critical downstream effectors of BMP signaling in human pulmonary arterial smooth muscle cells

    PubMed Central

    Yang, Jun; Li, Xiaohui; Li, Ying; Southwood, Mark; Ye, Lingying; Long, Lu; Al-Lamki, Rafia S.

    2013-01-01

    Bone morphogenetic protein type II receptor (BMPR-II) mutations are responsible for over 70% of cases of heritable pulmonary arterial hypertension (PAH). Loss of BMP signaling promotes pulmonary vascular remodeling via modulation of pulmonary artery smooth muscle cell (PASMC) proliferation. Id proteins (Id1–4) are major downstream transcriptional targets of BMP signaling. However, the impact of BMPR-II mutation on the expression of the range of Id proteins and the contribution of individual Id proteins to abnormal PASMC function remain unclear. Human PASMCs were used to determine the expression of Id proteins (Id1–4) by real-time PCR and immunoblotting. The BMP responses in control cells were compared with PASMCs harboring BMPR-II mutations and cells in which BMPR-II was knocked down by siRNA transfection. Id3 expression in pulmonary vessels was also investigated in BMPR-II mutant mice and in patients with heritable PAH. BMP4 and BMP6, but not BMP9, induced mRNA expression of Id1, Id2, and Id3. The BMP-stimulated induction of Id1 and Id3 was markedly reduced in BMPR-II mutant PASMCs and in control PASMCs following siRNA silencing of BMPR-II. Pulmonary arteries in BMPR-II mutant mice and patients with heritable PAH demonstrated reduced levels of Id3 compared with control subjects. Lentiviral overexpression of Id3 reduced cell cycle progression and inhibited proliferation of PASMCs. Lipopolysaccharide further reduced Id3 expression in mutant PASMCs. In conclusion, Id proteins, and particularly Id1 and Id3, are critical downstream effectors of BMP signaling in PASMCs. Loss of BMPR-II function reduces the induction of Id genes in PASMCs, Id1, and Id3 regulate the proliferation of PASMCs via cell cycle inhibition, an effect that may be exacerbated by inflammatory stimuli. PMID:23771884

  9. Interplay between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of the Hedgehog signaling pathway.

    PubMed

    Cheng, Peng; Wang, Yun-Feng; Li, Gang; Yang, Sheng-sheng; Liu, Che; Hu, Hao; Jin, Gang; Hu, Xian-Gui

    2016-01-01

    Menin, the product of the Men1 gene, which is frequently mutated in pancreatic neuroendocrine tumors, acts as a chromatin-remodeling factor to modulate the transcription of cell cycle regulators by interacting with histone modification factors. However, the function of menin and its underlying mechanisms in pancreatic ductal adenocarcinoma remain unknown. Here, we found that menin inhibited pancreatic cancer cell growth in vitro and in vivo and that its expression was gradually lost during pancreatic carcinogenesis. Menin overexpression significantly activated the expression of the cyclin-dependent kinase (CDK) inhibitors p18 and p27, accompanied with a decrease in DNA methylation levels of p18 and p27 promoters. Mechanistically, we found that interaction of menin with DNA methyltransferase 1 (Dnmt1) competitively pulled down Dnmt1 from p18 and p27 promoters, leading to the downregulation of DNA methylation levels. Moreover, menin expression was suppressed by Dnmt1 downstream of the Hedgehog signaling pathway, and menin overexpression strongly antagonized the promotion effect of hedgehog signaling on pancreatic cancer cell proliferation. Taken together, the interaction between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of Hedgehog pathways with complex mutual modulation networks, suggesting that the Hedgehog/Dnmt1/menin axis is a potential molecular target for pancreatic cancer therapy. PMID:26454216

  10. Baicalin inhibits toll-like receptor 2/4 expression and downstream signaling in rat experimental periodontitis.

    PubMed

    Sun, Jun-Yi; Li, Dong-Ling; Dong, Yan; Zhu, Chun-Hui; Liu, Jin; Li, Jue-Dan; Zhou, Tao; Gou, Jian-Zhong; Li, Ang; Zang, Wei-Jin

    2016-07-01

    Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis (P. gingivalis) for 4weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200mg/kg/day) or vehicle for 4weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1β, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis. PMID:27107801

  11. Clustering of monosialyl-Gb5 initiates downstream signalling events leading to invasion of MCF-7 breast cancer cells

    PubMed Central

    van Slambrouck, Severine; Steelant, Wim F. A.

    2006-01-01

    Invasion is a complex process controlled by secretion and activation of proteases, alteration of integrin levels and GSL (glycosphingolipid) patterns. Differential organization of GSLs with specific membrane proteins and signal transducers in GEMs (GSL-enriched microdomains), initiates signalling events to modify cellular phenotype. Although the GSL monosialyl-Gb5 has been linked with invasion, its functional role in invasion is poorly described and understood. To investigate this problem, we induced the invasion of human breast cancer cells and subsequently explored the underlying mechanism. In the present study, the invasion of human MCF-7 breast cancer cells is highly dependent on clustering of monosialyl-Gb5, and the subsequent activation of monosialyl-Gb5-associated focal adhesion kinase and cSrc in GEM leading to the downstream activation of extracellular-signal-regulated kinase (ERK). As a result, we observed increased expression levels and activity of matrix metalloproteinases-2 and -9, which correlated with decreased expression of integrins α1 and β1. Together these results suggest that the organization of crucial molecules in GEMs of MCF-7 cells is critical for their invasive properties. PMID:16995838

  12. Downstream activation of NF-κB in the EDA-A1/EDAR signalling in Sjögren's syndrome and its regulation by the ubiquitin-editing enzyme A20.

    PubMed

    Sisto, M; Barca, A; Lofrumento, D D; Lisi, S

    2016-05-01

    Sjögren's syndrome (SS) is an autoimmune disease and the second most common chronic systemic rheumatic disorder. Prevalence of primary SS in the general population has been estimated to be approximately 1-3%, whereas secondary SS has been observed in 10-20% of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma. Despite this, its exact aetiology and pathogenesis are largely unexplored. Nuclear factor-kappa B (NF-κB) signalling mechanisms provide central controls in SS, but how these pathways intersect the pathological features of this disease is unclear. The ubiquitin-editing enzyme A20 (tumour necrosis factor-α-induced protein 3, TNFAIP3) serves as a critical inhibitor on NF-κB signalling. In humans, polymorphisms in the A20 gene or a deregulated expression of A20 are often associated with several inflammatory disorders, including SS. Because A20 controls the ectodysplasin-A1 (EDA-A1)/ectodysplasin receptor (EDAR) signalling negatively, and the deletion of A20 results in excessive EDA1-induced NF-κB signalling, this work investigates the expression levels of EDA-A1 and EDAR in SS human salivary glands epithelial cells (SGEC) and evaluates the hypothesis that SS SGEC-specific deregulation of A20 results in excessive EDA1-induced NF-κB signalling in SS. Our approach, which combines the use of siRNA-mediated gene silencing and quantitative pathway analysis, was used to elucidate the role of the A20 target gene in intracellular EDA-A1/EDAR/NF-κB pathway in SS SGEC, holding significant promise for compound selection in drug discovery. PMID:26724675

  13. The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1

    PubMed Central

    Liu, Wensheng; Yue, Fei; Zheng, Minhua; Merlot, Angelica; Bae, Dong-Hun; Huang, Michael; Lane, Darius; Jansson, Patric; Liu, Goldie Yuan Lam; Richardson, Vera; Sahni, Sumit; Kalinowski, Danuta; Kovacevic, Zaklina; Richardson, Des. R.

    2015-01-01

    N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a “molecular switch” to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics. PMID:25860930

  14. Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

    SciTech Connect

    Inadera, Hidekuni Shimomura, Akiko; Tachibana, Shinjiro

    2009-02-20

    Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein {delta} expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor {gamma} expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-{alpha} did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.

  15. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase

    PubMed Central

    Leenders, Frauke; Möpert, Kristin; Schmiedeknecht, Anett; Santel, Ansgar; Czauderna, Frank; Aleku, Manuela; Penschuck, Silke; Dames, Sibylle; Sternberger, Maria; Röhl, Thomas; Wellmann, Axel; Arnold, Wolfgang; Giese, Klaus; Kaufmann, Jörg; Klippel, Anke

    2004-01-01

    Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K. PMID:15282551

  16. PTHrP promotes malignancy of human oral cancer cell downstream of the EGFR signaling

    SciTech Connect

    Yamada, Tamaki; Tsuda, Masumi; Ohba, Yusuke Kawaguchi, Hideaki; Totsuka, Yasunori; Shindoh, Masanobu

    2008-04-11

    Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.

  17. Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

    PubMed Central

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

    2015-01-01

    Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

  18. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor

    PubMed Central

    Delgado-Peraza, Francheska; Ahn, Kwang H.; Nogueras-Ortiz, Carlos; Mungrue, Imran N.; Mackie, Ken; Kendall, Debra A.

    2016-01-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  19. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor.

    PubMed

    Delgado-Peraza, Francheska; Ahn, Kwang H; Nogueras-Ortiz, Carlos; Mungrue, Imran N; Mackie, Ken; Kendall, Debra A; Yudowski, Guillermo A

    2016-06-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  20. Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux

    PubMed Central

    He, Yuan; Zeng, Melody Y.; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-01-01

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome

  1. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

    PubMed

    He, Yuan; Zeng, Melody Y; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-02-18

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to

  2. Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma.

    PubMed

    Ge, Xuecai; Milenkovic, Ljiljana; Suyama, Kaye; Hartl, Tom; Purzner, Teresa; Winans, Amy; Meyer, Tobias; Scott, Matthew P

    2015-01-01

    Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors. PMID:26371509

  3. Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma

    PubMed Central

    Ge, Xuecai; Milenkovic, Ljiljana; Suyama, Kaye; Hartl, Tom; Purzner, Teresa; Winans, Amy; Meyer, Tobias; Scott, Matthew P

    2015-01-01

    Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors. DOI: http://dx.doi.org/10.7554/eLife.07068.001 PMID:26371509

  4. An experimental demonstration for carrier reused bidirectional PON system with adaptive modulation DDO-OFDM downstream and QPSK upstream signals.

    PubMed

    Yan, Jhih-Heng; Chen, You-Wei; Shen, Kuan-Heng; Feng, Kai-Ming

    2013-11-18

    A light source centralized bidirectional passive optical network (PON) system based on multiband direct-detection optical orthogonal frequency division multiplexing (DDO-OFDM) downstream and quadrature phase-shift keying (QPSK) upstream is experimentally demonstrated. By introducing a simple optical single-side band (SSB) filter at the optical network unit (ONU), all the desired signal bands will be immune from the deleterious signal-signal beating interference (SSBI) noise with only single-end direct-detection scheme. An adaptive modulation configuration is employed to enhance the entire downstream throughput which results in a 150-Gbps downstream data rate with a single optical carrier. In the upstream direction, by recycling the clean downstream optical carrier, a 12.5 Gb/s QPSK format with coherent receiving mechanism in central office is adopted for better receiving sensitivity and dispersion tolerance. With the power enhancement by the long-reach PON architecture, the downstream splitting ratio can achieve as high as 1:1024. PMID:24514327

  5. AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer.

    PubMed

    Vasudevan, Krishna M; Barbie, David A; Davies, Michael A; Rabinovsky, Rosalia; McNear, Chontelle J; Kim, Jessica J; Hennessy, Bryan T; Tseng, Hsiuyi; Pochanard, Panisa; Kim, So Young; Dunn, Ian F; Schinzel, Anna C; Sandy, Peter; Hoersch, Sebastian; Sheng, Qing; Gupta, Piyush B; Boehm, Jesse S; Reiling, Jan H; Silver, Serena; Lu, Yiling; Stemke-Hale, Katherine; Dutta, Bhaskar; Joy, Corwin; Sahin, Aysegul A; Gonzalez-Angulo, Ana Maria; Lluch, Ana; Rameh, Lucia E; Jacks, Tyler; Root, David E; Lander, Eric S; Mills, Gordon B; Hahn, William C; Sellers, William R; Garraway, Levi A

    2009-07-01

    Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations. PMID:19573809

  6. miR-326 Is Downstream of Sonic Hedgehog Signaling and Regulates the Expression of Gli2 and Smoothened

    PubMed Central

    Jiang, Zhihua; Cushing, Leah; Lü, Jining

    2014-01-01

    Sonic hedgehog (Shh) is expressed and secreted from the embryonic lung epithelium and acts on the adjacent mesenchymal cells via its receptor Patched (Ptch)/Smoothened (Smo) and transcriptional effectors Gli proteins. Genetic studies showed that the Shh pathway plays critical roles in mouse lung development. However, little is known about microRNAs (miRNAs) downstream of Shh in embryonic lungs. Here we profiled miRNAs in embryonic lung cultures treated with cyclopamine, a specific Smo antagonist or with Smo agonist by next-generation of sequencing. We then performed functional screening to examine whether some of these miRNAs can modulate the induction of Gli-responsive luciferase by Shh treatment. These analyses revealed that expression of miR-326 and its host gene, Arrestin β1, is selectively enriched in embryonic lung mesenchymal cells and is specifically influenced by Shh activity. Furthermore, functional analyses showed that miR-326 acts as a negative modulator for Shh signaling by directly targeting Smo and Gli2. Together, these findings suggest a novel miR-326–negative feedback loop in regulating the activity of Shh signaling. PMID:24617895

  7. miR-326 is downstream of Sonic hedgehog signaling and regulates the expression of Gli2 and smoothened.

    PubMed

    Jiang, Zhihua; Cushing, Leah; Ai, Xingbin; Lü, Jining

    2014-08-01

    Sonic hedgehog (Shh) is expressed and secreted from the embryonic lung epithelium and acts on the adjacent mesenchymal cells via its receptor Patched (Ptch)/Smoothened (Smo) and transcriptional effectors Gli proteins. Genetic studies showed that the Shh pathway plays critical roles in mouse lung development. However, little is known about microRNAs (miRNAs) downstream of Shh in embryonic lungs. Here we profiled miRNAs in embryonic lung cultures treated with cyclopamine, a specific Smo antagonist or with Smo agonist by next-generation of sequencing. We then performed functional screening to examine whether some of these miRNAs can modulate the induction of Gli-responsive luciferase by Shh treatment. These analyses revealed that expression of miR-326 and its host gene, Arrestin β1, is selectively enriched in embryonic lung mesenchymal cells and is specifically influenced by Shh activity. Furthermore, functional analyses showed that miR-326 acts as a negative modulator for Shh signaling by directly targeting Smo and Gli2. Together, these findings suggest a novel miR-326-negative feedback loop in regulating the activity of Shh signaling. PMID:24617895

  8. An essential role for Grk2 in Hedgehog signalling downstream of Smoothened.

    PubMed

    Zhao, Zhonghua; Lee, Raymond Teck Ho; Pusapati, Ganesh V; Iyu, Audrey; Rohatgi, Rajat; Ingham, Philip W

    2016-05-01

    The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR. PMID:27113758

  9. Frequency of alcohol consumption in humans; the role of metabotropic glutamate receptors and downstream signaling pathways.

    PubMed

    Meyers, J L; Salling, M C; Almli, L M; Ratanatharathorn, A; Uddin, M; Galea, S; Wildman, D E; Aiello, A E; Bradley, B; Ressler, K; Koenen, K C

    2015-01-01

    Rodent models implicate metabotropic glutamate receptors (mGluRs) and downstream signaling pathways in addictive behaviors through metaplasticity. One way mGluRs can influence synaptic plasticity is by regulating the local translation of AMPA receptor trafficking proteins via eukaryotic elongation factor 2 (eEF2). However, genetic variation in this pathway has not been examined with human alcohol use phenotypes. Among a sample of adults living in Detroit, Michigan (Detroit Neighborhood Health Study; n = 788; 83% African American), 206 genetic variants across the mGluR-eEF2-AMPAR pathway (including GRM1, GRM5, HOMER1, HOMER2, EEF2K, MTOR, EIF4E, EEF2, CAMK2A, ARC, GRIA1 and GRIA4) were found to predict number of drinking days per month (corrected P-value < 0.01) when considered as a set (set-based linear regression conducted in PLINK). In addition, a CpG site located in the 3'-untranslated region on the north shore of EEF2 (cg12255298) was hypermethylated in those who drank more frequently (P < 0.05). Importantly, the association between several genetic variants within the mGluR-eEF2-AMPAR pathway and alcohol use behavior (i.e., consumption and alcohol-related problems) replicated in the Grady Trauma Project (GTP), an independent sample of adults living in Atlanta, Georgia (n = 1034; 95% African American), including individual variants in GRM1, GRM5, EEF2, MTOR, GRIA1, GRIA4 and HOMER2 (P < 0.05). Gene-based analyses conducted in the GTP indicated that GRM1 (empirical P < 0.05) and EEF2 (empirical P < 0.01) withstood multiple test corrections and predicted increased alcohol consumption and related problems. In conclusion, insights from rodent studies enabled the identification of novel human alcohol candidate genes within the mGluR-eEF2-AMPAR pathway. PMID:26101849

  10. Frequency of alcohol consumption in humans; the role of metabotropic glutamate receptors and downstream signaling pathways

    PubMed Central

    Meyers, J L; Salling, M C; Almli, L M; Ratanatharathorn, A; Uddin, M; Galea, S; Wildman, D E; Aiello, A E; Bradley, B; Ressler, K; Koenen, K C

    2015-01-01

    Rodent models implicate metabotropic glutamate receptors (mGluRs) and downstream signaling pathways in addictive behaviors through metaplasticity. One way mGluRs can influence synaptic plasticity is by regulating the local translation of AMPA receptor trafficking proteins via eukaryotic elongation factor 2 (eEF2). However, genetic variation in this pathway has not been examined with human alcohol use phenotypes. Among a sample of adults living in Detroit, Michigan (Detroit Neighborhood Health Study; n=788; 83% African American), 206 genetic variants across the mGluR–eEF2–AMPAR pathway (including GRM1, GRM5, HOMER1, HOMER2, EEF2K, MTOR, EIF4E, EEF2, CAMK2A, ARC, GRIA1 and GRIA4) were found to predict number of drinking days per month (corrected P-value <0.01) when considered as a set (set-based linear regression conducted in PLINK). In addition, a CpG site located in the 3′-untranslated region on the north shore of EEF2 (cg12255298) was hypermethylated in those who drank more frequently (P<0.05). Importantly, the association between several genetic variants within the mGluR–eEF2–AMPAR pathway and alcohol use behavior (i.e., consumption and alcohol-related problems) replicated in the Grady Trauma Project (GTP), an independent sample of adults living in Atlanta, Georgia (n=1034; 95% African American), including individual variants in GRM1, GRM5, EEF2, MTOR, GRIA1, GRIA4 and HOMER2 (P<0.05). Gene-based analyses conducted in the GTP indicated that GRM1 (empirical P<0.05) and EEF2 (empirical P<0.01) withstood multiple test corrections and predicted increased alcohol consumption and related problems. In conclusion, insights from rodent studies enabled the identification of novel human alcohol candidate genes within the mGluR–eEF2–AMPAR pathway. PMID:26101849

  11. Differential subcellular membrane recruitment of Src may specify its downstream signalling

    SciTech Connect

    Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah; D'Auria, Ludovic; Van Der Smissen, Patrick; Platek, Anna; Mettlen, Marcel; Caplanusi, Adrian; Hove, Marie-France van den; Tyteca, Donatienne; Courtoy, Pierre J.

    2008-04-15

    Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.

  12. KIR2DL4 differentially signals downstream functions in human NK cells through distinct structural modules.

    PubMed

    Miah, S M Shahjahan; Hughes, Tracey L; Campbell, Kerry S

    2008-03-01

    KIR2DL4 (2DL4) is a member of the killer cell Ig-like receptor (KIR) family in human NK cells. It can stimulate potent cytokine production and weak cytolytic activity in resting NK cells, but the mechanism for 2DL4-mediated signaling remains unclear. In this study we characterized the signaling pathways stimulated by 2DL4 engagement. In a human NK-like cell line, KHYG-1, cross-linking of 2DL4 activated MAPKs including JNK, ERK, and p38. Furthermore, 2DL4 cross-linking resulted in phosphorylation of IkappaB kinase beta (IKKbeta) and the phosphorylation and degradation of IkappaBalpha, which indicate activation of the classical NF-kappaB pathway. Engagement of 2DL4 was also shown to activate the transcription and translation of a variety of cytokine genes, including TNF-alpha, IFN-gamma, MIP1alpha, MIP1beta, and IL-8. Pharmacological inhibitors of JNK, MEK1/2 and p38, blocked IFN-gamma, IL-8, and MIP1alpha production, suggesting that MAPKs are regulating 2DL4-mediated cytokine production in a nonredundant manner. Activation of both p38 and ERK appear to be upstream of the stimulation of NF-kappaB. Mutation of a transmembrane arginine in 2DL4 to glycine (R/G mutant) abrogated FcepsilonRI-gamma association, as well as receptor-mediated cytolytic activity and calcium responses. Surprisingly, the R/G mutant still activated MAPKs and the NF-kappaB pathway and selectively stimulated the production of MIP1alpha, but not that of IFN-gamma or IL-8. In conclusion, we provide evidence that the activating functions of 2DL4 can be compartmentalized into two distinct structural modules: 1) through transmembrane association with FcepsilonRI-gamma; and 2) through another receptor domain independent of the transmembrane arginine. PMID:18292514

  13. Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells

    PubMed Central

    2013-01-01

    Background Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses. Bisphenol-A, used to manufacture plastic consumer products, and nonylphenol, a surfactant, are estrogenic by a variety of assays, including altering many intracellular signaling pathways; bisphenol-S is now used as a bisphenol-A substitute. All three compounds contaminate the environment globally. We previously showed that bisphenol-S, bisphenol-A, and nonylphenol alone rapidly activated several kinases at very low concentrations in the GH3/B6/F10 rat pituitary cell line. Methods For each assay we compared the response of individual xenoestrogens at environmentally relevant concentrations (10-15 -10-7 M), to their mixture effects on 10-9 M estradiol-induced responses. We used a medium-throughput plate immunoassay to quantify phosphorylations of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs). Cell numbers were assessed by crystal violet assay to compare the proliferative effects. Apoptosis was assessed by measuring caspase 8 and 9 activities via the release of the fluorescent product 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after a 1 min exposure to all individual and combinations of estrogens. Results Individual xenoestrogens elicited phospho-activation of ERK in a non-monotonic dose- (fM-nM) and mostly oscillating time-dependent (2.5-60 min) manner. When multiple xenoestrogens were combined with nM estradiol, the physiologic estrogen’s response was attenuated. Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose–response. Estradiol and all xenoestrogen compounds induced cell proliferation individually, while the mixtures of these compounds with estradiol suppressed proliferation below that of the

  14. The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways.

    PubMed

    Kovacevic, Zaklina; Menezes, Sharleen V; Sahni, Sumit; Kalinowski, Danuta S; Bae, Dong-Hun; Lane, Darius J R; Richardson, Des R

    2016-01-15

    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

  15. A simple feature construction method for predicting upstream/downstream signal flow in human protein-protein interaction networks

    PubMed Central

    Mei, Suyu; Zhu, Hao

    2015-01-01

    Signaling pathways play important roles in understanding the underlying mechanism of cell growth, cell apoptosis, organismal development and pathways-aberrant diseases. Protein-protein interaction (PPI) networks are commonly-used infrastructure to infer signaling pathways. However, PPI networks generally carry no information of upstream/downstream relationship between interacting proteins, which retards our inferring the signal flow of signaling pathways. In this work, we propose a simple feature construction method to train a SVM (support vector machine) classifier to predict PPI upstream/downstream relations. The domain based asymmetric feature representation naturally embodies domain-domain upstream/downstream relations, providing an unconventional avenue to predict the directionality between two objects. Moreover, we propose a semantically interpretable decision function and a macro bag-level performance metric to satisfy the need of two-instance depiction of an interacting protein pair. Experimental results show that the proposed method achieves satisfactory cross validation performance and independent test performance. Lastly, we use the trained model to predict the PPIs in HPRD, Reactome and IntAct. Some predictions have been validated against recent literature. PMID:26648121

  16. Transglutaminase inhibition protects against oxidative stress-induced neuronal death downstream of pathological ERK activation

    PubMed Central

    Basso, Manuela; Berlin, Jill; Li, Xia; Sleiman, Sama F.; Ko, Brendan; Haskew-Layton, Renee; Kim, Eunhee; Antonyak, Marc A.; Cerione, Richard A.; Iismaa, Siiri E.; Willis, Dianna; Cho, Sunghee; Ratan, Rajiv R.

    2012-01-01

    Molecular deletion of transglutaminase 2 (TG2) has been shown to improve function and survival in a host of neurological conditions including stroke, Huntington’s disease, and Parkinson’s disease. However, unifying schemes by which these crosslinking or polyaminating enzymes participate broadly in neuronal death have yet to be presented. Unexpectedly, we found that in addition to TG2, TG1 gene expression level is significantly induced following stroke in vivo or due to oxidative stress in vitro. Forced expression of TG1 or TG2 proteins is sufficient to induce neuronal death in Rattus novergicus cortical neurons in vitro. Accordingly, molecular deletion of TG2 alone is insufficient to protect Mus musculus neurons from oxidative death. By contrast, structurally diverse inhibitors used at concentrations that inhibit TG1 and TG2 simultaneously are neuroprotective. These small molecules inhibit increases in neuronal transamidating activity induced by oxidative stress; they also protect neurons downstream of pathological ERK activation when added well after the onset of the death stimulus. Together, these studies suggest that multiple TG isoforms, not only TG2, participate in oxidative stress-induced cell death signaling; and that isoform non-selective inhibitors of TG will be most efficacious in combating oxidative death in neurological disorders. PMID:22573678

  17. Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells

    PubMed Central

    Spampatti, Matilde; Vlotides, George; Spöttl, Gerald; Maurer, Julian; Göke, Burkhard; Auernhammer, Christoph J

    2014-01-01

    AIM: To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro. METHODS: Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student’s t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P < 0.05. RESULTS: Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P < 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P < 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P < 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1

  18. Selective inhibition of EGFR downstream signaling reverses the irradiation-enhanced migration of HNSCC cells

    PubMed Central

    Schuettler, Dominik; Piontek, Guido; Wirth, Markus; Haller, Bernhard; Reiter, Rudolf; Brockhoff, Gero; Pickhard, Anja

    2015-01-01

    Irradiation, which is one of the standard therapies used to treat squamous cell carcinoma of the head and neck (HNSCC), has been linked to enhanced tumor migration in carcinomas. In this study, we demonstrated that irradiation induced the phosphorylation of AKT, p38 MAPK and ERK. The combined activation of these pathways caused inactivation of GSK3β kinase, resulting in enhanced tumor cell migration. Here, we describe that the exclusive and specific inhibition of just one of the aforementioned key signaling molecules is sufficient to restore GSK3β activity and to reduce radiation-induced migration in HNSCC. These data indicate that pharmacological inhibition of pathways targeting GSK3β could decrease radiation-induced cell migration in HNSCC and thus potentially reduce metastasis and locoregional recurrence in patients. PMID:26609474

  19. Twist induces epithelial-mesenchymal transition and cell motility in breast cancer via ITGB1-FAK/ILK signaling axis and its associated downstream network.

    PubMed

    Yang, Jiajia; Hou, Yixuan; Zhou, Mingli; Wen, Siyang; Zhou, Jian; Xu, Liyun; Tang, Xi; Du, Yan-e; Hu, Ping; Liu, Manran

    2016-02-01

    Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-β, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC-MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM-receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity Pathways Analysis showed that integrin β1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells. PMID:26693891

  20. The β-catenin Axis Integrates Multiple Signals Downstream From RET/PTC and Leads to Cell Proliferation

    PubMed Central

    Castellone, Maria Domenica; De Falco, Valentina; Rao, Deva Magendra; Bellelli, Roberto; Muthu, Magesh; Basolo, Fulvio; Fusco, Alfredo; Gutkind, J. Silvio; Santoro, Massimo

    2009-01-01

    RET/PTC (RET/papillary thyroid carcinoma) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase domain with protein dimerization motifs encoded by heterologous genes. Here we show that RET/PTC stimulates the β-catenin pathway. By stimulating PI3K/AKT and Ras/ERK, RET/PTC promotes GSK3β phosphorylation, thereby reducing GSK3β-mediated N-terminal β-catenin (Ser33/Ser37/Thr41) phosphorylation. In addition, RET/PTC physically interacts with β-catenin, and increases its phosphotyrosine content. The increased free pool of S/T(nonphospho)/Y(phospho)β-catenin is stabilized as a result of the reduced binding affinity for the Axin/GSK3β complex and activates the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor. Moreover, through the ERK pathway, RET/PTC stimulates cAMP-responsive element binding protein (CREB) phosphorylation and promotes the formation of a β-catenin-CREB-CBP/p300 transcriptional complex. Transcriptional complexes containing β-catenin are recruited to the cyclin D1 promoter and a cyclin D1 gene promoter reporter is active in RET/PTC expressing cells. Silencing of β-catenin by siRNA inhibits proliferation of RET/PTC transformed PC thyrocytes, whereas a constitutively active form of β-catenin stimulates autonomous proliferation of thyroid cells. Thus, multiple signaling events downstream from RET/PTC converge on β-catenin to stimulate cell proliferation. PMID:19223551

  1. PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis.

    PubMed

    Martin, Katherine; Pritchett, James; Llewellyn, Jessica; Mullan, Aoibheann F; Athwal, Varinder S; Dobie, Ross; Harvey, Emma; Zeef, Leo; Farrow, Stuart; Streuli, Charles; Henderson, Neil C; Friedman, Scott L; Hanley, Neil A; Piper Hanley, Karen

    2016-01-01

    Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis. PMID:27535340

  2. PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

    PubMed Central

    Martin, Katherine; Pritchett, James; Llewellyn, Jessica; Mullan, Aoibheann F.; Athwal, Varinder S.; Dobie, Ross; Harvey, Emma; Zeef, Leo; Farrow, Stuart; Streuli, Charles; Henderson, Neil C.; Friedman, Scott L.; Hanley, Neil A.; Piper Hanley, Karen

    2016-01-01

    Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis. PMID:27535340

  3. Nicotine enhances invasion and metastasis of human colorectal cancer cells through the nicotinic acetylcholine receptor downstream p38 MAPK signaling pathway.

    PubMed

    Xiang, Tao; Fei, Rushan; Wang, Zhe; Shen, Zhonglei; Qian, Jing; Chen, Wenbin

    2016-01-01

    Nicotine as a cigarette component is an established risk factor for colorectal cancer tumorigenesis. The downstream signaling pathways of nicotinic acetylcholine receptors (nAchRs) are believed to be responsible for the cellular effects. In the present study, we evaluated the effects and novel mechanisms for nicotine on the capacity for colorectal cancer cell invasion and metastasis. LOVO and SW620 colorectal cancer cells were stimulated with nicotine in vitro. A Transwell chamber model was applied to detect the capacity for tumor cell invasion. Assays for gelatin zymography and western blotting were applied to detect the activity and expression of metastasis-related matrix metalloproteinases (MMPs), respectively. Signal transduction was assessed by immunoblotting for the phosphorylation of relevant signal molecules and the application of pharmaceutical inhibitors. We showed that nicotine increased LOVO and SW620 colorectal cancer cell invasion along with enhanced activity and expression of MMP-1, -2 and -9. Nicotine increased phosphorylation of p38, ERK, Akt and PI3K p85 but had no effect on phosphorylation of JNK, or NF-κB. Of the pharmaceutical inhibitors of U0126 (ERK1/2 inhibitor), LY294002 (Akt activation inhibitor), SB239063 (p38 MAPK activation inhibitor) and hexamethonium (Hex) (nAchRs inhibitor), the cellular and molecular effects were reduced by the applications of SB239063 and Hex. We concluded that nicotine stimulates the invasion and metastasis of colon cancer cells in vitro via activation of the nAchRs and the p38 MAPK downstream signaling pathway. Therefore, p38 MAPK may have potential as a therapeutic target for smoking-related human colorectal cancer metastasis. PMID:26530054

  4. Distinct Signal Transduction Pathways Downstream of the (P)RR Revealed by Microarray and ChIP-chip Analyses

    PubMed Central

    Zaade, Daniela; Schmitz, Jennifer; Benke, Eileen; Klare, Sabrina; Seidel, Kerstin; Kirsch, Sebastian; Goldin-Lang, Petra; Zollmann, Frank S.; Unger, Thomas; Funke-Kaiser, Heiko

    2013-01-01

    The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)RŔs signal transduction pathways in cardiovascular disease and tumorigenesis. PMID:23469216

  5. Downstream Effect of Ramping Neuronal Activity through Synapses with Short-Term Plasticity.

    PubMed

    Wei, Wei; Wang, Xiao-Jing

    2016-04-01

    Ramping neuronal activity refers to spiking activity with a rate that increases quasi-linearly over time. It has been observed in multiple cortical areas and is correlated with evidence accumulation processes or timing. In this work, we investigated the downstream effect of ramping neuronal activity through synapses that display short-term facilitation (STF) or depression (STD). We obtained an analytical result for a synapse driven by deterministic linear ramping input that exhibits pure STF or STD and numerically investigated the general case when a synapse displays both STF and STD. We show that the analytical deterministic solution gives an accurate description of the averaging synaptic activation of many inputs converging onto a postsynaptic neuron, even when fluctuations in the ramping input are strong. Activation of a synapse with STF shows an initial cubical increase with time, followed by a linear ramping similar to a synapse without STF. Activation of a synapse with STD grows in time to a maximum before falling and reaching a plateau, and this steady state is independent of the slope of the ramping input. For a synapse displaying both STF and STD, an increase in the depression time constant from a value much smaller than the facilitation time constant τ(F) to a value much larger than τ(F) leads to a transition from facilitation dominance to depression dominance. Therefore, our work provides insights into the impact of ramping neuronal activity on downstream neurons through synapses that display short-term plasticity. In a perceptual decision-making process, ramping activity has been observed in the parietal and prefrontal cortices, with a slope that decreases with task difficulty. Our work predicts that neurons downstream from such a decision circuit could instead display a firing plateau independent of the task difficulty, provided that the synaptic connection is endowed with short-term depression. PMID:26890350

  6. Roles of EGFR and KRAS and their downstream signaling pathways in pancreatic cancer and pancreatic cancer stem cells.

    PubMed

    Fitzgerald, Timothy L; Lertpiriyapong, Kvin; Cocco, Lucio; Martelli, Alberto M; Libra, Massimo; Candido, Saverio; Montalto, Giuseppe; Cervello, Melchiorre; Steelman, Linda; Abrams, Stephen L; McCubrey, James A

    2015-09-01

    Pancreatic cancer is currently the fourth most common cancer, is increasing in incidence and soon will be the second leading cause of cancer death in the USA. This is a deadly malignancy with an incidence that approximates the mortality with 44,000 new cases and 36,000 deaths each year. Surgery, although only modestly successful, is the only curative option. However, due the locally aggressive nature and early metastasis, surgery can be performed on less than 20% of patients. Cytotoxic chemotherapy is palliative, has significant toxicity and improves survival very little. Thus new treatment paradigms are needed desperately. Due to the extremely high frequency of KRAS gene mutations (>90%) detected in pancreatic cancer patients, the roles of the epidermal growth factor receptor (EGFR), Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTORC1/GSK-3 pathways have been investigated in pancreatic cancer for many years. Constitutively active Ras can activate both of these pathways and there is cross talk between Ras and EGFR which is believed to be important in driving metastasis. Mutant KRAS may also drive the expression of GSK-3 through Raf/MEK/ERK-mediated effects on GSK-3 transcription. GSK-3 can then regulate the expression of NF-kappaB which is important in modulating pancreatic cancer chemoresistance. While the receptors and many downstream signaling molecules have been identified and characterized, there is still much to learn about these pathways and how their deregulation can lead to cancer. Multiple inhibitors to EGFR, PI3K, mTOR, GSK-3, Raf, MEK and hedgehog (HH) have been developed and are being evaluated in various cancers. Current research often focuses on the role of these pathways in cancer stem cells (CSC), with the goal to identify sites where therapeutic resistance may develop. Relatively novel fields of investigation such as microRNAs and drugs used for other diseases e.g., diabetes, (metformin) and malaria (chloroquine) have provided new information about

  7. Bitter, sweet and umami taste receptors and downstream signaling effectors: Expression in embryonic and growing chicken gastrointestinal tract.

    PubMed

    Cheled-Shoval, Shira L; Druyan, Shelly; Uni, Zehava

    2015-08-01

    Taste perception is a crucial biological mechanism affecting food and water choices and consumption in the animal kingdom. Bitter taste perception is mediated by a G-protein-coupled receptor (GPCR) family-the taste 2 receptors (T2R)-and their downstream proteins, whereas sweet and umami tastes are mediated by the GPCR family -taste 1 receptors (T1R) and their downstream proteins. Taste receptors and their downstream proteins have been identified in extra-gustatory tissues in mammals, such as the lungs and gastrointestinal tract (GIT), and their GIT activation has been linked with different metabolic and endocrinic pathways in the GIT. The chicken genome contains three bitter taste receptors termed ggTas2r1, ggTas2r2, and ggTas2r7, and the sweet/umami receptors ggTas1r1 and ggTas1r3, but it lacks the sweet receptor ggTas1r2. The aim of this study was to identify and determine the expression of genes related to taste perception in the chicken GIT, both at the embryonic stage and in growing chickens. The results of this study demonstrate for the first time, using real-time PCR, expression of the chicken taste receptor genes ggTas2r1, ggTas2r2, ggTas2r7, ggTas1r1, and ggTas1r3 and of their downstream protein-encoding genes TRPM5, α-gustducin, and PLCβ2 in both gustatory tissues-the palate and tongue, and extra-gustatory tissues-the proventriculus, duodenum, jejunum, ileum, cecum, and colon of embryonic day 19 (E19) and growing (21 d old) chickens. Expression of these genes suggests the involvement of taste pathways for sensing carbohydrates, amino acids and bitter compounds in the chicken GIT. PMID:26049797

  8. Binding Selectivity of Abaloparatide for PTH-Type-1-Receptor Conformations and Effects on Downstream Signaling.

    PubMed

    Hattersley, Gary; Dean, Thomas; Corbin, Braden A; Bahar, Hila; Gardella, Thomas J

    2016-01-01

    The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone. PMID:26562265

  9. Polycomb repressive complex PRC2 regulates Xenopus retina development downstream of Wnt/β-catenin signaling

    PubMed Central

    Aldiri, Issam; Moore, Kathryn B.; Hutcheson, David A.; Zhang, Jianmin; Vetter, Monica L.

    2013-01-01

    The histone methyltransferase complex PRC2 controls key steps in developmental transitions and cell fate choices; however, its roles in vertebrate eye development remain unknown. Here, we report that in Xenopus, PRC2 regulates the progression of retinal progenitors from proliferation to differentiation. We show that the PRC2 core components are enriched in retinal progenitors and downregulated in differentiated cells. Knockdown of the PRC2 core component Ezh2 leads to reduced retinal progenitor proliferation, in part due to upregulation of the Cdk inhibitor p15Ink4b. In addition, although PRC2 knockdown does not alter eye patterning, retinal progenitor gene expression or expression of the neural competence factor Sox2, it does cause suppression of proneural bHLH gene expression, indicating that PRC2 is crucial for the initiation of neural differentiation in the retina. Consistent with this, knocking down or blocking PRC2 function constrains the generation of most retinal neural cell types and promotes a Müller glial cell fate decision. We also show that Wnt/β-catenin signaling acting through the receptor Frizzled 5, but independent of Sox2, regulates expression of key PRC2 subunits in the developing retina. This is consistent with a role for this pathway in coordinating proliferation and the transition to neurogenesis in the Xenopus retina. Our data establish PRC2 as a regulator of proliferation and differentiation during eye development. PMID:23739135

  10. Quantitative phosphoproteomic analysis of signaling downstream of the prostaglandin e2/g-protein coupled receptor in human synovial fibroblasts: potential antifibrotic networks.

    PubMed

    Gerarduzzi, Casimiro; He, QingWen; Antoniou, John; Di Battista, John A

    2014-11-01

    The Prostaglandin E2 (PGE2) signaling mechanism within fibroblasts is of growing interest as it has been shown to prevent numerous fibrotic features of fibroblast activation with limited evidence of downstream pathways. To understand the mechanisms of fibroblasts producing tremendous amounts of PGE2 with autocrine effects, we apply a strategy of combining a wide-screening of PGE2-induced kinases with quantitative phosphoproteomics. Our large-scale proteomic approach identified a PKA signal transmitted through phosphorylation of its substrates harboring the R(R/X)X(S*/T*) motif. We documented 115 substrates, of which 72 had 89 sites with a 2.5-fold phosphorylation difference in PGE2-treated cells than in untreated cells, where approximately half of such sites were defined as being novel. They were compiled by networking software to focus on highlighted activities and to associate them with a functional readout of fibroblasts. The substrates were associated with a variety of cellular functions including cytoskeletal structures (migration/motility), regulators of G-protein coupled receptor function, protein kinases, and transcriptional/translational regulators. For the first time, we extended the PGE2 pathway into an elaborate network of interconnecting phosphoproteins, providing vital information to a once restricted signalosome. These data provide new insights into eicosanoid-initiated cell signaling with regards to the regulation of fibroblast activation and the identification of new targets for evidenced-based pharmacotherapy against fibrosis. PMID:25223752

  11. Symmetric 10 Gb/s wavelength reused bidirectional RSOA based WDM-PON with DPSK modulated downstream and OFDM modulated upstream signals

    NASA Astrophysics Data System (ADS)

    Choudhury, Pallab K.; Khan, Tanvir Zaman

    2016-08-01

    A 10 Gb/s bidirectional wavelength division multiplexing passive optical network (WDM-PON) with reflective semiconductor optical amplifier (RSOA) based colorless optical network unit (ONU) is proposed and analyzed for next generation gigabit class optical access network. Differential phase shift keying (DPSK) modulated signal is used in downstream and further reused as a seeding wavelength for upstream data modulation. By exploiting the constant envelope property of DPSK seed signal, the re-modulation noise in upstream receiver is effectively minimized without employing any constraint on extinction ratio of downstream signal. Orthogonal frequency division multiplexing (OFDM) signal is used in upstream transmission to overcome the limited bandwidth (∼1 GHz) response of RSOA remodulation. The results show that the proposed 10 Gb/s symmetric WDM-PON can achieve good performance over 25 km fiber transmission with error free operation in downstream and bit error rate (BER) lower than forward error correction (FEC) limit in upstream.

  12. Signal focusing through active transport

    NASA Astrophysics Data System (ADS)

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing—faster and more precise signaling—are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  13. Molecular Steps in the Immune Signaling Pathway Evoked by Plant Elicitor Peptides: Ca2+-Dependent Protein Kinases, Nitric Oxide, and Reactive Oxygen Species Are Downstream from the Early Ca2+ Signal1[OPEN

    PubMed Central

    Ma, Yi; Zhao, Yichen; Walker, Robin K.; Berkowitz, Gerald A.

    2013-01-01

    Endogenous plant elicitor peptides (Peps) can act to facilitate immune signaling and pathogen defense responses. Binding of these peptides to the Arabidopsis (Arabidopsis thaliana) plasma membrane-localized Pep receptors (PEPRs) leads to cytosolic Ca2+ elevation, an early event in a signaling cascade that activates immune responses. This immune response includes the amplification of signaling evoked by direct perception of pathogen-associated molecular patterns by plant cells under assault. Work included in this report further characterizes the Pep immune response and identifies new molecular steps in the signal transduction cascade. The PEPR coreceptor BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 contributes to generation of the Pep-activated Ca2+ signal and leads to increased defense gene expression and resistance to a virulent bacterial pathogen. Ca2+-dependent protein kinases (CPKs) decode the Ca2+ signal, also facilitating defense gene expression and enhanced resistance to the pathogen. Nitric oxide and reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species generation (due to the function of Respiratory Burst Oxidase Homolog proteins D and F) are also involved downstream from the Ca2+ signal in the Pep immune defense signal transduction cascade, as is the case with BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 and CPK5, CPK6, and CPK11. These steps of the pathogen defense response are required for maximal Pep immune activation that limits growth of a virulent bacterial pathogen in the plant. We find a synergism between function of the PEPR and Flagellin Sensing2 receptors in terms of both nitric oxide and reactive oxygen species generation. Presented results are also consistent with the involvement of the secondary messenger cyclic GMP and a cyclic GMP-activated Ca2+-conducting channel in the Pep immune signaling pathway. PMID:24019427

  14. Transcription factor network downstream of protease activated receptors (PARs) modulating mouse bladder inflammation

    PubMed Central

    Saban, Ricardo; Simpson, Cindy; Davis, Carole A; Dozmorov, Igor; Maier, Julie; Fowler, Ben; Ihnat, Michael A; Hurst, Robert E; Wershil, Barry K; Saban, Marcia R

    2007-01-01

    Background All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. Methods For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kitw/Kitw-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. Results TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kitw/Kitw-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kitw/Kitw-v mice. Conclusion This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders. PMID:17705868

  15. Effect of intervention in the diacylglycerol‑protein kinase C signaling pathway on JNK1 expression and its downstream signaling in diabetic cardiomyopathy.

    PubMed

    Liu, Xiaoliang; Qi, Fang; Wu, Wei

    2014-03-01

    This study aimed to investigate the expression of signaling molecules, such as c‑Jun N‑terminal kinase 1 (JNK1) and insulin receptor substrate 1 (IRS1), in the myocardium of diabetic rats following intervention in the diacylglycerol‑protein kinase C (DAG‑PKC) signal transduction pathway. The rats were divided into three groups, the diabetic model, control and breviscapine‑treated diabetes (intervention) group. Following modeling and drug treatment, hematoxylin and eosin (HE) and Masson staining and electron microscopy were used to observe the pathological changes in the rat myocardium. The expression of PKC‑β2, JNK1, and IRS1 was assessed in rat myocardium by immunohistochemistry and quantitative polymerase chain reaction (qPCR). The expression levels of PKC‑β2, JNK1, phosphorylated JNK (p‑JNK) and IRS1 in the diabetic model group were significantly higher than those in the control group. Furthermore, compared with the diabetic model group, expression levels of PKC‑β2, JNK1, p‑JNK and IRS1 were significantly reduced following intervention in the DAG‑PKC signal transduction pathway. The DAG‑PKC pathway may affect downstream signaling through JNK1 (the common signal point of the G‑protein receptor pathway and insulin receptor pathway at the cell membrane) to result in the occurrence and development of diabetic cardiomyopathy (DCM). The series of signal points DAG‑PKC‑JNK1‑IRS1‑Akt/PKB‑mTOR‑p70S6K1 is a potential pathway for inducing DCM by DAG‑PKC signal transduction. Enhanced expression of JNK1, p‑JNK and IRS1 may accelerate diabetic myocardial fibrosis. PMID:24435585

  16. PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met-addicted tumor cells.

    PubMed

    Kherrouche, Zoulika; Monte, Didier; Werkmeister, Elisabeth; Stoven, Luc; De Launoit, Yvan; Cortot, Alexis B; Tulasne, David; Chotteau-Lelievre, Anne

    2015-11-01

    Various solid tumors including lung or gastric carcinomas display aberrant activation of the Met receptor which correlates with aggressive phenotypes and poor prognosis. Although downstream signaling of Met is well described, its integration at the transcriptional level is poorly understood. We demonstrate here that in cancer cells harboring met gene amplification, inhibition of Met activity with tyrosine kinase inhibitors or specific siRNA drastically decreased expression of ETV1, ETV4 and ETV5, three transcription factors constituting the PEA3 subgroup of the ETS family, while expression of the other members of the family were less or not affected. Similar link between Met activity and PEA3 factors expression was found in lung cancer cells displaying resistance to EGFR targeted therapy involving met gene amplification. Using silencing experiments, we demonstrate that the PEA3 factors are required for efficient migration and invasion mediated by Met, while other biological responses such as proliferation or unanchored growth remain unaffected. PEA3 overexpression or silencing revealed that they participated in the regulation of the MMP2 target gene involved in extracellular matrix remodeling. Our results demonstrated that PEA3-subgroup transcription factors are key players of the Met signaling integration involved in regulation of migration and invasiveness. PMID:26238631

  17. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling.

    PubMed

    Tee, Andrew R; Fingar, Diane C; Manning, Brendan D; Kwiatkowski, David J; Cantley, Lewis C; Blenis, John

    2002-10-15

    Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products hamartin and tuberin, respectively. Here, we show that hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets. PMID:12271141

  18. NRG1 and KITL Signal Downstream of Retinoic Acid in the Germline to Support Soma-Free Syncytial Growth of Differentiating Spermatogonia

    PubMed Central

    Chapman, Karen M.; Medrano, Gerardo A.; Chaudhary, Jaideep; Hamra, F. Kent

    2015-01-01

    Defined culture systems supporting spermatogonial differentiation will provide experimental platforms to study spermatogenesis. However, germline-intrinsic signaling mechanisms sufficient to support spermatogonial differentiation without somatic cells remain largely undefined. Here, we analyzed EGF superfamily receptor and ligand diversity in rat testis cells, and delineated germline-intrinsic signaling via an ERBB3 co-transducer, ERBB2, as essential for retinoic acid-induced syncytial growth by differentiating spermatogonia. Like the ERBB2/3 agonist NRG1, we found KIT Ligand (KITL) robustly supported spermatogonial differentiation without serum or somatic cells. ERBB2 inhibitors failed to disrupt KITL-dependent spermatogonial development, and, KITL prevented ERBB3-deficient spermatogonial degeneration upon differentiation. Thus, we report NRG1 and KITL activate alternative pathways downstream of retinoic acid signaling in the germline that are essential for stem cells to undergo pre-meiotic steps of spermatogenesis in culture. Robust serum/soma-free spermatogonial differentiation opens new doors to study mammalian germ cell biology in culture, which will facilitate the discovery of spermatogenic factors that can drive meiotic progression in vitro. PMID:26500786

  19. Suppression or Induction of Apoptosis by Opposing Pathways Downstream from Calcium-Activated Calcineurin

    NASA Astrophysics Data System (ADS)

    Lotem, Joseph; Kama, Rachel; Sachs, Leo

    1999-10-01

    Ca2+-mobilizing compounds such as the Ca2+ ionophore A23187 or the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin can suppress or induce apoptosis in the same cells. The use of different calcineurin inhibitors has shown that both suppression and induction of apoptosis by the Ca2+-mobilizing compounds were mediated by calcineurin activation. Ca2+-mobilizing compounds activated p38 and p44/42 mitogen-activated protein kinases (MAPKs). Induction of apoptosis by the Ca2+-mobilizing compounds was suppressed by an inhibitor of p38 MAPK but not by an inhibitor of p44/42 MAPK. These MAPK inhibitors did not suppress apoptosis induction by wild-type p53 or by withdrawal of IL-6 from IL-6-dependent cells that are mediated by calcineurin-independent pathways. These MAPK inhibitors also did not affect the ability of Ca2+-mobilizing compounds to suppress apoptosis. The results indicate that (i) Ca2+-mobilizing compounds activate different and opposing pathways that diverge downstream from calcineurin activation that can either suppress or induce apoptosis in the same cells; (ii) p38 MAPK but not p44/42 MAPK is involved in induction of apoptosis but not in its suppression by the Ca2+-mobilizing compounds; and (iii) neither p38 nor p44/42 MAPKs mediate induction of apoptosis by some calcineurin-independent pathways.

  20. Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59[C][W][OA

    PubMed Central

    Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C.; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P.; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C.M.; Pieterse, Corné M.J.

    2013-01-01

    Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59. PMID:23435661

  1. Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice

    PubMed Central

    Zhao, Jing; Wang, Yanqing; Wu, Gencheng; Mi, Wenli

    2015-01-01

    Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways. PMID:26067287

  2. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5

    PubMed Central

    Bardita, Cristina; Predescu, Dan N.; Sha, Fei; Patel, Monal; Balaji, Ganesh; Predescu, Sanda A.

    2015-01-01

    Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5–Smad–SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5–mSos–Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2MAPK signaling that is significant for proliferative signaling and abnormal growth. PMID:25720380

  3. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5.

    PubMed

    Bardita, Cristina; Predescu, Dan N; Sha, Fei; Patel, Monal; Balaji, Ganesh; Predescu, Sanda A

    2015-04-15

    Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5-Smad-SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5-mSos-Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2(MAPK) signaling that is significant for proliferative signaling and abnormal growth. PMID:25720380

  4. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    PubMed

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  5. New crossroads for potential therapeutic intervention in cancer - intersections between CDCP1, EGFR family members and downstream signaling pathways

    PubMed Central

    Hooper, John D.

    2016-01-01

    Signaling pathways regulated by the receptor CDCP1 play central roles in promoting cancer and in mediating resistance to chemo- and targeted-therapies. In this perspective we briefly summarize these findings as well as data demonstrating poorer outcomes for several malignancies that exhibit elevated CDCP1 expression. Promising data from preclinical studies suggest that CDCP1 targeted agents, including therapeutic antibodies, could be useful in the treatment of cancer patients selected on the basis of activation of CDCP1 and its signaling partners including EGFR, HER2, Met and Src. PMID:26973855

  6. mTOR signaling pathway is inhibited downstream of the cyclophilin D-mediated mitochondrial permeability transition in honokiol-triggered regulated necrosis.

    PubMed

    Tian, Wei; Xiong, Jieni; Zhu, Saisa; Xu, Dong; Shen, Hong; Deng, Yongchuan

    2016-04-01

    Honokiol (HNK) is a pharmacologically active small molecule that is isolated from the traditional Chinese medicinal herb, houpu. It may induce diversified types of regulated cell death, which are dependent on different cell types and varying concentrations of therapeutic agent. We previously reported that HNK triggers a cyclophilin D (CypD)-mediated regulated necrosis in various cell lines at certain concentrations (two‑fold higher than its half maximal inhibitory concentration). Subsequent study revealed that HNK induced cell death transition from early apoptosis to regulated necrosis in parallel with the increase of HNK dose. In the current study, a lower concentration of HNK (30 µg/ml) than previously reported also induced simplex CypD‑mediated mitochondrial permeability transition (MPT)‑associated regulated necrosis in the HEK‑293 human embryonic kidney cell line. HNK, at concentration of 30 µg/ml, induced necrotic cell death in HEK‑293 cells, which was demonstrated by positive staining for propidium iodide. No DNA ladder patterns or apoptotic bodies were detected in cells that underwent this type of necrotic cell death. Caspase‑8 and ‑3 were not activated during the process of HNK‑induced necrosis. In addition, pan‑caspase inhibitor, z‑VAD‑fmk and receptor‑interacting protein 1 inhibitor, necrostatin‑1 did not inhibit HNK‑induced necrosis. However, CypD inhibitor, cyclosporin A (CsA), blocked HNK‑induced necrosis. These findings indicate that 30 µg/ml HNK induced simplex CypD-mediated MPT‑associated regulated necrosis in HEK‑293 cells. Furthermore, the findings demonstrated that during HNK-triggered regulated necrosis the mammalian target of rapamycin (mTOR) signaling pathway is also inhibited. Pretreatment with CsA, therefore, inhibits HNK‑triggered regulated necrosis and reverses dephosphorylation of Akt, eIF4E‑binding protein 1 and S6 kinase. This indicated that the mTOR signaling pathway is effective

  7. Receptor tyrosine kinases: mechanisms of activation and signaling

    PubMed Central

    Hubbard, Stevan R.; Miller, W. Todd

    2008-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands — mainly growth factors — play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell. PMID:17306972

  8. Effect of Redox Balance Alterations on Cellular Localization of LAT and Downstream T-Cell Receptor Signaling Pathways

    PubMed Central

    Gringhuis, Sonja I.; Papendrecht-van der Voort, Ellen A. M.; Leow, Angela; Levarht, E. W. Nivine; Breedveld, Ferdinand C.; Verweij, Cornelis L.

    2002-01-01

    The integral membrane protein linker for activation of T cells (LAT) is a central adapter protein in the T-cell receptor (TCR)-mediated signaling pathways. The cellular localization of LAT is extremely sensitive to intracellular redox balance alterations. Reduced intracellular levels of the antioxidant glutathione (GSH), a hallmark of chronic oxidative stress, resulted in the membrane displacement of LAT, abrogated TCR-mediated signaling and consequently hyporesponsiveness of T lymphocytes. The membrane displacement of LAT is accompanied by a considerable difference in the mobility of LAT upon native and nonreducing denaturing polyacrylamide gel electrophoresis analysis, a finding indicative of a conformational change. Targeted mutation of redox-sensitive cysteine residues within LAT created LAT mutants which remain membrane anchored under conditions of chronic oxidative stress. The expression of redox-insensitive LAT mutants allows for restoration of TCR-mediated signal transduction, whereas CD28-mediated signaling pathways remained impaired. These results are indicative that the membrane displacement of LAT as a result of redox balance alterations is a consequence of a conformational change interfering with the insertion of LAT into the plasma membrane. Conclusively, the data suggest a role for LAT as a crucial intermediate in the sensitivity of TCR signaling and hence T lymphocytes toward chronic oxidative stress. PMID:11756537

  9. Ganglioside GM2 mediates migration of tumor cells by interacting with integrin and modulating the downstream signaling pathway.

    PubMed

    Kundu, Manjari; Mahata, Barun; Banerjee, Avisek; Chakraborty, Sohini; Debnath, Shibjyoti; Ray, Sougata Sinha; Ghosh, Zhumur; Biswas, Kaushik

    2016-07-01

    The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor. PMID:27066976

  10. ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination.

    PubMed

    Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K; Ucher, Anna J; Leus, Niek G J; Ogilvie, Colin; Lou, Zhenkun; Schrader, Carol E; Stavnezer, Janet

    2014-05-15

    Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sμ DSBs accumulate as they lack a recombination partner. PMID:24729610

  11. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress.

    PubMed

    Yoshida, Takuya; Fujita, Yasunari; Maruyama, Kyonoshin; Mogami, Junro; Todaka, Daisuke; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2015-01-01

    Under osmotic stress conditions such as drought and high salinity, the plant hormone abscisic acid (ABA) plays important roles in stress-responsive gene expression mainly through three bZIP transcription factors, AREB1/ABF2, AREB2/ABF4 and ABF3, which are activated by SNF1-related kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). However, since the three AREB/ABFs are crucial, but not exclusive, for the SnRK2-mediated gene expression, transcriptional pathways governed by SRK2D/E/I are not fully understood. Here, we show that a bZIP transcription factor, ABF1, is a functional homolog of AREB1, AREB2 and ABF3 in ABA-dependent gene expression in Arabidopsis. Despite lower expression levels of ABF1 than those of the three AREB/ABFs, the areb1 areb2 abf3 abf1 mutant plants displayed increased sensitivity to drought and decreased sensitivity to ABA in primary root growth compared with the areb1 areb2 abf3 mutant. Genome-wide transcriptome analyses revealed that expression of downstream genes of SRK2D/E/I, which include many genes functioning in osmotic stress responses and tolerance such as transcription factors and LEA proteins, was mostly impaired in the quadruple mutant. Thus, these results indicate that the four AREB/ABFs are the predominant transcription factors downstream of SRK2D/E/I in ABA signalling in response to osmotic stress during vegetative growth. PMID:24738645

  12. Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: implications for Alzheimer's disease intervention.

    PubMed

    Zhao, Lixia; Teter, Bruce; Morihara, Takashi; Lim, Giselle P; Ambegaokar, Surendra S; Ubeda, Oliver J; Frautschy, Sally A; Cole, Greg M

    2004-12-01

    Insulin-degrading enzyme (IDE) is one of the proteins that has been demonstrated to play a key role in degrading beta-amyloid (Abeta) monomer in vitro and in vivo, raising the possibility of upregulating IDE as an approach to reduce Abeta. Little is known, however, about the cellular and molecular regulation of IDE protein. Because one of the main functions of IDE is to degrade insulin, we hypothesized that there is a negative feedback mechanism whereby stimulation of insulin receptor-mediated signaling upregulates IDE to prevent chronic activation of the pathway. We show that treatment of primary hippocampal neurons with insulin increased IDE protein levels by approximately 25%. Insulin treatment also led to phosphatidylinositol-3 (PI3) kinase activation evidenced by Akt phosphorylation, which was blocked by PI3 kinase inhibitors, wortmannin and LY 294002. Inhibition of PI3 kinase abolished the IDE upregulation by insulin, indicating a cause-effect relationship between insulin signaling and IDE upregulation. Further support for this link was provided by the findings that deficient insulin signaling (decreased PI3 kinase subunit P85) was correlated with reduced IDE in Alzheimer's disease (AD) brains and in Tg2576 Swedish amyloid precursor protein transgenic mice fed a safflower oil-enriched ("Bad") diet used to accelerate pathogenesis. Consistent with IDE function in the degradation of Abeta monomer, the IDE decrease in the Bad diet-fed Tg2576 mice was associated with increased Abeta monomer levels. These in vitro and in vivo analyses validate the use of enhanced CNS insulin signaling as a potential strategy for AD intervention to correct the IDE defects occurring in AD. PMID:15590928

  13. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    PubMed Central

    Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C.; Paulus, Christina

    2016-01-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  14. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response.

    PubMed

    Harwardt, Thomas; Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C; Nevels, Michael; Paulus, Christina

    2016-07-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  15. Involvement of PI3K/Akt Signaling Pathway and Its Downstream Intracellular Targets in the Antidepressant-Like Effect of Creatine.

    PubMed

    Cunha, Mauricio P; Budni, Josiane; Ludka, Fabiana K; Pazini, Francis L; Rosa, Julia Macedo; Oliveira, Ágatha; Lopes, Mark W; Tasca, Carla I; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

    2016-07-01

    Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 μg/mouse, icv, PI3K inhibitor), ZnPP (10 μg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 μg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 μg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition. PMID:25943184

  16. Cleavage of E-cadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling.

    PubMed

    Solanas, Guiomar; Cortina, Carme; Sevillano, Marta; Batlle, Eduard

    2011-09-01

    The formation and maintenance of complex organs requires segregation of distinct cell populations into defined territories (that is, cell sorting) and the establishment of boundaries between them. Here we have investigated the mechanism by which Eph/ephrin signalling controls the compartmentalization of cells in epithelial tissues. We show that EphB/ephrin-B signalling in epithelial cells regulates the formation of E-cadherin-based adhesions. EphB receptors interact with E-cadherin and with the metalloproteinase ADAM10 at sites of adhesion and their activation induces shedding of E-cadherin by ADAM10 at interfaces with ephrin-B1-expressing cells. This process results in asymmetric localization of E-cadherin and, as a consequence, in differences in cell affinity between EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in the intestine of mice results in a lack of compartmentalization of Paneth cells within the crypt stem cell niche, a defect that phenocopies that of EphB3-null mice. These results provide important insights into the regulation of cell migration in the intestinal epithelium and may help in the understanding of the nature of the cell sorting process in other epithelial tissues where Eph-ephrin interactions play a central role. PMID:21804545

  17. Cellular phosphatases facilitate combinatorial processing of receptor-activated signals

    PubMed Central

    Kumar, Dhiraj; Dua, Raina; Srikanth, Ravichandran; Jayaswal, Shilpi; Siddiqui, Zaved; Rao, Kanury VS

    2008-01-01

    Background Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response. Results To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR) under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors. Conclusion Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile. PMID:18798986

  18. Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia

    PubMed Central

    Zhu, Qiu-Ju; Kong, Fan-Shu; Xu, Hao; Wang, Yi; Du, Cai-Ping; Sun, Chang-Cheng; Liu, Yong; Li, Ting; Hou, Xiao-Yu

    2014-01-01

    Although kainate receptors play important roles in ischemic stroke, the molecular mechanisms underlying postischemic regulation of kainate receptors remain unclear. In this study we demonstrate that Src family kinases contribute to the potentiation of kainate receptor function. Brain ischemia and reperfusion induce rapid and sustained phosphorylation of the kainate receptor subunit GluK2 by Src in the rat hippocampus, implicating a critical role for Src-mediated GluK2 phosphorylation in ischemic brain injury. The NMDA and kainate receptors are involved in the tyrosine phosphorylation of GluK2. GluK2 binds to Src, and the tyrosine residue at position 590 (Y590) on GluK2 is a major site of phosphorylation by Src kinases. GluK2 phosphorylation at Y590 is responsible for increases in whole-cell currents and calcium influx in response to transient kainate stimulation. In addition, GluK2 phosphorylation at Y590 facilitates the endocytosis of GluK2 subunits, and the activation of JNK3 and its substrate c-Jun after long-term kainate treatment. Thus, Src phosphorylation of GluK2 plays an important role in the opening of kainate receptor channels and downstream proapoptosis signaling after brain ischemia. The present study reveals an additional mechanism for the regulation of GluK2-containing kainate receptors by Src family kinases, which may be of pathological significance in ischemic stroke. PMID:25201974

  19. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    PubMed

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells. PMID:27306974

  20. Geniposide plays an anti-inflammatory role via regulating TLR4 and downstream signaling pathways in lipopolysaccharide-induced mastitis in mice.

    PubMed

    Song, Xiaojing; Zhang, Wen; Wang, Tiancheng; Jiang, Haichao; Zhang, Zecai; Fu, Yunhe; Yang, Zhengtao; Cao, Yongguo; Zhang, Naisheng

    2014-10-01

    Geniposide is a medicine isolated from Gardenia jasminoides Ellis, which is a traditional Chinese herb that is widely used in Asia for the treatment of inflammation, brain diseases, and hepatic disorders. Mastitis is a highly prevalent and important infectious disease. In this study, we used a lipopolysaccharide (LPS)-induced mouse mastitis model and LPS-stimulated primary mouse mammary epithelial cells (mMECs) to explore the anti-inflammatory effect and the mechanism of action of geniposide. Using intraductal injection of LPS as a mouse model of mastitis, we found that geniposide significantly reduced the infiltration of inflammatory cells and downregulated the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). To further investigate the anti-inflammatory mechanism, we used LPS-stimulated mMECs as an in vitro mastitis model. The results of enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) showed that geniposide inhibited the expression of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Western blot analysis demonstrated that geniposide could suppress the phosphorylation of inhibitory kappa B (IκBα), nuclear factor-κB (NF-κB), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Geniposide also inhibited the expression of toll-like receptor 4 (TLR4) in the LPS-stimulated mMECs. In conclusion, geniposide exerted its anti-inflammatory effect by regulating TLR4 expression, which affected the downstream NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Thus, geniposide may be a potential drug for mastitis therapy. PMID:24771071

  1. Unique Effects of Acute Aripiprazole Treatment on the Dopamine D2 Receptor Downstream cAMP-PKA and Akt-GSK3β Signalling Pathways in Rats

    PubMed Central

    Pan, Bo; Chen, Jiezhong; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2015-01-01

    Aripiprazole is a wide-used antipsychotic drug with therapeutic effects on both positive and negative symptoms of schizophrenia, and reduced side-effects. Although aripiprazole was developed as a dopamine D2 receptor (D2R) partial agonist, all other D2R partial agonists that aimed to mimic aripiprazole failed to exert therapeutic effects in clinic. The present in vivo study aimed to investigate the effects of aripiprazole on the D2R downstream cAMP-PKA and Akt-GSK3β signalling pathways in comparison with a D2R antagonist – haloperidol and a D2R partial agonist – bifeprunox. Rats were injected once with aripiprazole (0.75mg/kg, i.p.), bifeprunox (0.8mg/kg, i.p.), haloperidol (0.1mg/kg, i.p.) or vehicle. Five brain regions – the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), ventral tegmental area (VTA) and substantia nigra (SN) were collected. The protein levels of PKA, Akt and GSK3β were measured by Western Blotting; the cAMP levels were examined by ELISA tests. The results showed that aripiprazole presented similar acute effects on PKA expression to haloperidol, but not bifeprunox, in the CPU and VTA. Additionally, aripiprazole was able to increase the phosphorylation of GSK3β in the PFC, NAc, CPu and SN, respectively, which cannot be achieved by bifeprunox and haloperidol. These results suggested that acute treatment of aripiprazole had differential effects on the cAMP-PKA and Akt-GSK3β signalling pathways from haloperidol and bifeprunox in these brain areas. This study further indicated that, by comparison with bifeprunox, the unique pharmacological profile of aripiprazole may be attributed to the relatively lower intrinsic activity at D2R. PMID:26162083

  2. FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

    PubMed

    Fan, Qipeng; Cai, Qingchun; Xu, Yan

    2015-09-29

    Lysophosphatidic acid (LPA), a prototypical ligand for G protein coupled receptors, and Forkhead box protein M1 (FOXM1), a transcription factor that regulates expression of a wide array of genes involved in cancer initiation and progression, are two important oncogenic signaling molecules in human epithelial ovarian cancers (EOC). We conducted in vitro mechanistic studies using pharmacological inhibitors, genetic forms of the signaling molecules, and RNAi-mediated gene knock-down to uncover the molecular mechanisms of how these two molecules interact in EOC cells. Additionally, in vivo mouse studies were performed to confirm the functional involvement of FOXM1 in EOC tumor formation and progression. We show for the first time that LPA up-regulates expression of active FOXM1 splice variants in a time- and dose-dependent manner in the human EOC cell lines OVCA433, CAOV3, and OVCAR5. Gi-PI3K-AKT and G12/13-Rho-YAP signaling pathways were both involved in the LPA receptor (LPA1-3) mediated up-regulation of FOXM1 at the transcriptional level. In addition, down-regulation of FOXM1 in CAOV3 xenografts significantly reduced tumor and ascites formation, metastasis, and expression of FOXM1 target genes involved in cell proliferation, migration, or invasion. Collectively, our data link the oncolipid LPA, the oncogene YAP, and the central regulator of cell proliferation/mutagenesis FOXM1 in EOC cells. Moreover, these results provide further support for the importance of these pathways as potential therapeutic targets in EOC. PMID:26299613

  3. Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF.

    PubMed

    Bernard, Monique; Dieudé, Mélanie; Yang, Bing; Hamelin, Katia; Underwood, Katy; Hébert, Marie-Josée

    2014-01-01

    Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation. PMID:25495560

  4. Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF

    PubMed Central

    Bernard, Monique; Dieudé, Mélanie; Yang, Bing; Hamelin, Katia; Underwood, Katy; Hébert, Marie-Josée

    2015-01-01

    Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation. PMID:25495560

  5. Hydrogen sulfide acts as a downstream signal molecule in salicylic acid-induced heat tolerance in maize (Zea mays L.) seedlings.

    PubMed

    Li, Zhong-Guang; Xie, Lin-Run; Li, Xiao-Juan

    2015-04-01

    Salicylic acid (SA), 2-hydroxy benzoic acid, is a small phenolic compound with multifunction that is involved in plant growth, development, and the acquisition of stress tolerance. In recent years, hydrogen sulfide (H2S) has been found to have similar functions, but cross talk between SA and H2S in the acquisition of heat tolerance is not clear. In this study, pretreatment of maize seedlings with SA improved the survival percentage of seedlings under heat stress, indicating that SA pretreatment could improve the heat tolerance of maize seedlings. In addition, treatment with SA enhanced the activity of L-cysteine desulfhydrase (L-DES), a key enzyme in H2S biosynthesis, which in turn induced accumulation of endogenous H2S. Interestingly, SA-induced heat tolerance was enhanced by addition of NaHS, a H2S donor, but weakened by specific inhibitors of H2S biosynthesis DL-propargylglycine (PAG) and its scavenger hydroxylamine (HT). Furthermore, pretreatment with paclobutrazol (PAC) and 2-aminoindan-2-phosphonic acid (AIP), inhibitors of SA biosynthesis, had no significant effect on NaHS-induced heat tolerance of maize seedlings. Similarly, significant change in the activities of phenylalanine ammonia lyase (PAL) and benzoic-acid-2-hydroxylase (BA2H), the key enzymes in SA biosynthesis, and the content of endogenous SA, was not observed in maize seedlings by NaHS treatment. All of the above-mentioned results suggest that SA pretreatment could improve the heat tolerance of maize seedlings, and H2S might be a novel downstream signal molecule in SA-induced heat tolerance. PMID:25727780

  6. Receptor-like cytoplasmic kinase MARIS functions downstream of CrRLK1L-dependent signaling during tip growth.

    PubMed

    Boisson-Dernier, Aurélien; Franck, Christina Maria; Lituiev, Dmytro S; Grossniklaus, Ueli

    2015-09-29

    Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRI(R240C) mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth. PMID:26378127

  7. Receptor-like cytoplasmic kinase MARIS functions downstream of CrRLK1L-dependent signaling during tip growth

    PubMed Central

    Boisson-Dernier, Aurélien; Franck, Christina Maria; Lituiev, Dmytro S.; Grossniklaus, Ueli

    2015-01-01

    Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRIR240C mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth. PMID:26378127

  8. Power Budget Analysis of Colorless Hybrid WDM/TDM-PON Scheme Using Downstream DPSK and Re-modulated Upstream OOK Data Signals

    NASA Astrophysics Data System (ADS)

    Khan, Yousaf; Afridi, Muhammad Idrees; Khan, Ahmed Mudassir; Rehman, Waheed Ur; Khan, Jahanzeb

    2014-09-01

    Hybrid wavelength-division multiplexed/time-division multiplexed passive optical access networks (WDM/TDM-PONs) combine the advance features of both WDM and TDM PONs to provide a cost-effective access network solution. We demonstrate and analyze the transmission performances and power budget issues of a colorless hybrid WDM/TDM-PON scheme. A 10-Gb/s downstream differential phase shift keying (DPSK) and remodulated upstream on/off keying (OOK) data signals are transmitted over 25 km standard single mode fiber. Simulation results show error free transmission having adequate power margins in both downstream and upstream transmission, which prove the applicability of the proposed scheme to future passive optical access networks. The power budget confines both the PON splitting ratio and the distance between the Optical Line Terminal (OLT) and Optical Network Unit (ONU).

  9. Trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling in HER2-positive breast cancer

    PubMed Central

    Lion, Maëva; Harlé, Alexandre; Salleron, Julia; Ramacci, Carole; Campone, Mario; Merlin, Jean-Louis

    2016-01-01

    Human epidermal growth factor 2 (HER2) is overexpressed in 15–20% of breast carcinomas. The overexpression of HER2 was previously associated with a poor prognosis until the development of the first anti-HER2 therapy, trastuzumab, which drastically improves the prognosis of HER2-overexpressing breast cancers. However, its mechanism of action remains not fully understood. Several studies have proposed that the behavior and mechanism of action of trastuzumab may be drastically altered in vitro and in vivo. The present study assesses the ability of trastuzumab to inhibit the phosphorylation of the key-proteins of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin and Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways in vitro, in breast cancer cell lines and in tumor biopsies obtained from patients treated with trastuzumab preoperative monotherapy as part of the Unicancer GEP04 RADHER phase II clinical trial. HER2-positive SKBR3 and HER2-negative MCF-7 cell lines were exposed to trastuzumab for 72 h. In total, 41 patients received trastuzumab alone for 6 weeks of preoperative treatment. Biopsies were collected at the baseline and at surgery. A total of 19 pairs of associated baseline and surgery tumor specimens were eligible for protein extraction and comparative phosphoprotein expression analysis, prior to and subsequent to treatment. The expression of phosphoproteins was quantitatively assessed using a multiplex immunoassay. In the SKBR3 cell line, a statistically significant decrease of the expression level of phosphorylated (p-)AKT, p-ribosomal protein S6 kinase B1, p-extracellular signal regulated kinase 1/2 and p-mitogen-activated protein kinase kinase 1 was observed after exposure to trastuzumab. In contrast, no statistically significant variations for levels expression of these phosphoproteins were observed in patients following treatment. The lack of downregulation of PI3K and MAPK pathways could probably

  10. Gbx2 is required for the morphogenesis of the mouse inner ear: a downstream candidate of hindbrain signaling.

    PubMed

    Lin, Zhengshi; Cantos, Raquel; Patente, Maria; Wu, Doris K

    2005-05-01

    Gbx2 is a homeobox-containing transcription factor that is related to unplugged in Drosophila. In mice, Gbx2 and Otx2 negatively regulate each other to establish the mid-hindbrain boundary in the neural tube. Here, we show that Gbx2 is required for the development of the mouse inner ear. Absence of the endolymphatic duct and swelling of the membranous labyrinth are common features in Gbx2-/- inner ears. More severe mutant phenotypes include absence of the anterior and posterior semicircular canals, and a malformed saccule and cochlear duct. However, formation of the lateral semicircular canal and its ampulla is usually unaffected. These inner ear phenotypes are remarkably similar to those reported in kreisler mice, which have inner ear defects attributed to defects in the hindbrain. Based on gene expression analyses, we propose that activation of Gbx2 expression within the inner ear is an important pathway whereby signals from the hindbrain regulate inner ear development. In addition, our results suggest that Gbx2 normally promotes dorsal fates such as the endolymphatic duct and semicircular canals by positively regulating genes such as Wnt2b and Dlx5. However, Gbx2 promotes ventral fates such as the saccule and cochlear duct, possibly by restricting Otx2 expression. PMID:15829521

  11. Assembly-driven activation of the AIM2 foreign-dsDNA sensor provides a polymerization template for downstream ASC

    NASA Astrophysics Data System (ADS)

    Morrone, Seamus R.; Matyszewski, Mariusz; Yu, Xiong; Delannoy, Michael; Egelman, Edward H.; Sohn, Jungsan

    2015-07-01

    AIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2PYD) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2PYD oligomers define the filamentous structure, and the helical symmetry of the AIM2PYD filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2PYD is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

  12. Analysis of signal transduction pathways during anoxia exposure in a marine snail: a role for p38 MAP kinase and downstream signaling cascades.

    PubMed

    Larade, Kevin; Storey, Kenneth B

    2006-01-01

    The responses of members of the three main MAPK families (ERK, JNK/SAPK, p38 MAPK), as well as selected peripheral pathways, were examined in hepatopancreas of the marine periwinkle, Littorina littorea, to determine if anoxia exposure influenced the total protein content or the phosphorylation status of any key components. The content of active phospho-p38 MAPK was 2-fold higher in hepatopancreas from anoxic snails relative to controls. A 1.7-fold increase in the amount of phospho-Hsp27 and a 1.3-fold increase in phospho-CREB correlated well with the changes in p38 MAPK phosphorylation. Activation of these factors via p38 MAPK may be vital to the reorganization of metabolic responses to anoxia in hepatopancreas. No changes in components of the JNK/SAPK and ERK pathways occurred and transcription factors involved in lipid metabolism did not appear to be affected by anoxia. The present analysis of a variety of signaling pathways has implicated the p38 MAPK pathway as a key anoxia-responsive signal transduction pathway in L. littorea. PMID:16326124

  13. Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

    PubMed Central

    Meininger, Isabel; Griesbach, Richard A.; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C.; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-01-01

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation. PMID:27068814

  14. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway.

    PubMed

    Wu, Xu; Walker, John; Zhang, Jie; Ding, Sheng; Schultz, Peter G

    2004-09-01

    Previously, a small molecule, purmorphamine, was identified that selectively induces osteogenesis in multipotent mesenchymal progenitor cells. In order to gain insights into the mechanism of action of purmorphamine, high-density oligonucleotide microarrays were used to profile gene expression in multipotent mesenchymal progenitor cells treated with either purmorphamine or bone morphogenetic protein-4 (BMP-4). In contrast to BMP-4 treatment, purmorphamine activates the Hedgehog (Hh) signaling pathway, resulting in the up- and downregulation of its downstream target genes, including Gli1 and Patched. Moreover, the known Hh signaling antagonists, cyclopamine and forskolin, completely block the osteogenesis and Glimediated transcription induced by purmorphamine. These results demonstrate that purmorphamine is a small molecule agonist of Hedgehog signaling, and it may ultimately be useful in the treatment of bone-related disease and neurodegenerative disease. PMID:15380183

  15. Reg-2, A Downstream Signaling Protein in the Ciliary Neurotrophic Factor Survival Pathway, Alleviates Experimental Autoimmune Encephalomyelitis.

    PubMed

    Jiang, Hong; Tian, Ke-Wei; Zhang, Fan; Wang, Beibei; Han, Shu

    2016-01-01

    Ciliary neurotrophic factor (CNTF), originally described as a neurocytokine that could support the survival of neurons, has been recently found to alleviate demyelination, prevent axon loss, and improve functional recovery in a rat model of acute experimental autoimmune encephalomyelitis (EAE). However, poor penetration into the brain parenchyma and unfavorable side effects limit the utility of CNTF. Here, we evaluated the therapeutic potential of a protein downstream of CNTF, regeneration gene protein 2 (Reg-2). Using multiple morphological, molecular biology, and electrophysiological methods to assess neuroinflammation, axonal loss, demyelination, and functional impairment, we observed that Reg-2 and CNTF exert similar effects in the acute phase of EAE. Both treatments attenuated axonal loss and demyelination, improved neuronal survival, and produced functional improvement. With a smaller molecular weight and improved penetration into the brain parenchyma, Reg-2 may be a useful substitute for CNTF therapy in EAE and multiple sclerosis (MS). PMID:27242448

  16. Reg-2, A Downstream Signaling Protein in the Ciliary Neurotrophic Factor Survival Pathway, Alleviates Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Jiang, Hong; Tian, Ke-Wei; Zhang, Fan; Wang, Beibei; Han, Shu

    2016-01-01

    Ciliary neurotrophic factor (CNTF), originally described as a neurocytokine that could support the survival of neurons, has been recently found to alleviate demyelination, prevent axon loss, and improve functional recovery in a rat model of acute experimental autoimmune encephalomyelitis (EAE). However, poor penetration into the brain parenchyma and unfavorable side effects limit the utility of CNTF. Here, we evaluated the therapeutic potential of a protein downstream of CNTF, regeneration gene protein 2 (Reg-2). Using multiple morphological, molecular biology, and electrophysiological methods to assess neuroinflammation, axonal loss, demyelination, and functional impairment, we observed that Reg-2 and CNTF exert similar effects in the acute phase of EAE. Both treatments attenuated axonal loss and demyelination, improved neuronal survival, and produced functional improvement. With a smaller molecular weight and improved penetration into the brain parenchyma, Reg-2 may be a useful substitute for CNTF therapy in EAE and multiple sclerosis (MS). PMID:27242448

  17. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

    PubMed

    Liu, Jessica A J; Wu, Ming-Hoi; Yan, Carol H; Chau, Bolton K H; So, Henry; Ng, Alvis; Chan, Alan; Cheah, Kathryn S E; Briscoe, James; Cheung, Martin

    2013-02-19

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

  18. Wnt signaling and the activation of myogenesis in mammals.

    PubMed

    Cossu, G; Borello, U

    1999-12-15

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has opened up the possibility of linking these signaling events to the activation of myogenic regulatory factor genes such as Myf5 and MyoD and functionally related genes such as Pax3. Here we focus on the role of Wnts, their putative receptors Frizzled and the soluble antagonist Frzb1 in regulating mammalian myogenesis. Although it is becoming evident that the signaling downstream of Frizzled receptors is much more complex than anticipated, it is conceivable that it may lead to transcriptional activation of Myf5 and MyoD and to initiation of myogenesis. However, the fact that both Wnts and Sonic hedgehog have a strong effect on cell proliferation and survival suggests that they may contribute to the overall process of myogenesis by a combination of these different biological activities. PMID:10601008

  19. Wnt signaling and the activation of myogenesis in mammals.

    PubMed Central

    Cossu, G; Borello, U

    1999-01-01

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has opened up the possibility of linking these signaling events to the activation of myogenic regulatory factor genes such as Myf5 and MyoD and functionally related genes such as Pax3. Here we focus on the role of Wnts, their putative receptors Frizzled and the soluble antagonist Frzb1 in regulating mammalian myogenesis. Although it is becoming evident that the signaling downstream of Frizzled receptors is much more complex than anticipated, it is conceivable that it may lead to transcriptional activation of Myf5 and MyoD and to initiation of myogenesis. However, the fact that both Wnts and Sonic hedgehog have a strong effect on cell proliferation and survival suggests that they may contribute to the overall process of myogenesis by a combination of these different biological activities. PMID:10601008

  20. LLM-Domain B-GATA Transcription Factors Promote Stomatal Development Downstream of Light Signaling Pathways in Arabidopsis thaliana Hypocotyls.

    PubMed

    Klermund, Carina; Ranftl, Quirin L; Diener, Julia; Bastakis, Emmanouil; Richter, René; Schwechheimer, Claus

    2016-03-01

    Stomata are pores that regulate the gas and water exchange between the environment and aboveground plant tissues, including hypocotyls, leaves, and stems. Here, we show that mutants of Arabidopsis thaliana LLM-domain B-GATA genes are defective in stomata formation in hypocotyls. Conversely, stomata formation is strongly promoted by overexpression of various LLM-domain B-class GATA genes, most strikingly in hypocotyls but also in cotyledons. Genetic analyses indicate that these B-GATAs act upstream of the stomata formation regulators SPEECHLESS(SPCH), MUTE, and SCREAM/SCREAM2 and downstream or independent of the patterning regulators TOO MANY MOUTHS and STOMATAL DENSITY AND DISTRIBUTION1 The effects of the GATAs on stomata formation are light dependent but can be induced in dark-grown seedlings by red, far-red, or blue light treatments. PHYTOCHROME INTERACTING FACTOR(PIF) mutants form stomata in the dark, and in this genetic background, GATA expression is sufficient to induce stomata formation in the dark. Since the expression of the LLM-domain B-GATAs GNC(GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED) and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 as well as that of SPCH is red light induced but the induction of SPCH is compromised in a GATA gene mutant background, we hypothesize that PIF- and light-regulated stomata formation in hypocotyls is critically dependent on LLM-domain B-GATA genes. PMID:26917680

  1. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  2. Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a

    PubMed Central

    Liu, Chunqiao; Lin, Chen; Gao, Chun; May-Simera, Helen; Swaroop, Anand; Li, Tiansen

    2014-01-01

    ABSTRACT Planar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six “core PCP” components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. We recently demonstrated a role of Prickle1, a core PCP molecule in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, we generated three mutant alleles in mice. We show that the complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Curiously, Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a or Ror2 gene defects, prompting us to explore an association of Prickle1 with the Wnt pathway. We show that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. Our studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome. PMID:25190059

  3. The transcriptional repressor Blimp-1 acts downstream of BMP signaling to generate primordial germ cells in the cricket Gryllus bimaculatus.

    PubMed

    Nakamura, Taro; Extavour, Cassandra G

    2016-01-15

    Segregation of the germ line from the soma is an essential event for transmission of genetic information across generations in all sexually reproducing animals. Although some well-studied systems such as Drosophila and Xenopus use maternally inherited germ determinants to specify germ cells, most animals, including mice, appear to utilize zygotic inductive cell signals to specify germ cells during later embryogenesis. Such inductive germ cell specification is thought to be an ancestral trait of Bilateria, but major questions remain as to the nature of an ancestral mechanism to induce germ cells, and how that mechanism evolved. We previously reported that BMP signaling-based germ cell induction is conserved in both the mouse Mus musculus and the cricket Gryllus bimaculatus, which is an emerging model organism for functional studies of induction-based germ cell formation. In order to gain further insight into the functional evolution of germ cell specification, here we examined the Gryllus ortholog of the transcription factor Blimp-1 (also known as Prdm1), which is a widely conserved bilaterian gene known to play a crucial role in the specification of germ cells in mice. Our functional analyses of the Gryllus Blimp-1 ortholog revealed that it is essential for Gryllus primordial germ cell development, and is regulated by upstream input from the BMP signaling pathway. This functional conservation of the epistatic relationship between BMP signaling and Blimp-1 in inductive germ cell specification between mouse and cricket supports the hypothesis that this molecular mechanism regulated primordial germ cell specification in a last common bilaterian ancestor. PMID:26786211

  4. Activators of G Protein Signaling in the Kidney

    PubMed Central

    2015-01-01

    Heterotrimeric G proteins play a crucial role in regulating signal processing to maintain normal cellular homeostasis, and subtle perturbations in its activity can potentially lead to the pathogenesis of renal disorders or diseases. Cell-surface receptors and accessory proteins, which normally modify and organize the coupling of individual G protein subunits, contribute to the regulation of heterotrimeric G protein activity and their convergence and/or divergence of downstream signaling initiated by effector systems. Activators of G protein signaling (AGS) are a family of accessory proteins that intervene at multiple distinct points during the activation–inactivation cycle of G proteins, even in the absence of receptor stimulation. Perturbations in the expression of individual AGS proteins have been reported to modulate signal transduction pathways in a wide array of diseases and disorders within the brain, heart, immune system, and more recently, the kidney. This review will provide an overview of the expression profile, localization, and putative biologic role of the AGS family in the context of normal and diseased states of the kidney. PMID:25628392

  5. Spatial Regulation and the Rate of Signal Transduction Activation

    PubMed Central

    Batada, Nizar N; Shepp, Larry A; Siegmund, David O; Levitt, Michael

    2006-01-01

    Of the many important signaling events that take place on the surface of a mammalian cell, activation of signal transduction pathways via interactions of cell surface receptors is one of the most important. Evidence suggests that cell surface proteins are not as freely diffusible as implied by the classic fluid mosaic model and that their confinement to membrane domains is regulated. It is unknown whether these dynamic localization mechanisms function to enhance signal transduction activation rate or to minimize cross talk among pathways that share common intermediates. To determine which of these two possibilities is more likely, we derive an explicit equation for the rate at which cell surface membrane proteins interact based on a Brownian motion model in the presence of endocytosis and exocytosis. We find that in the absence of any diffusion constraints, cell surface protein interaction rate is extremely high relative to cytoplasmic protein interaction rate even in a large mammalian cell with a receptor abundance of a mere two hundred molecules. Since a larger number of downstream signaling events needs to take place, each occurring at a much slower rate than the initial activation via association of cell surface proteins, we conclude that the role of co-localization is most likely that of cross-talk reduction rather than coupling efficiency enhancement. PMID:16699596

  6. Phytocomponent 4-hydroxy-3-methoxycinnamaldehyde ablates T-cell activation by targeting protein kinase C-θ and its downstream pathways.

    PubMed

    Akber, Uroos; Na, Bo-Ra; Ko, Yoo-Seung; Ko, You-Seung; Lee, Hyun-Su; Kim, Hye-Ran; Kwon, Min-Sung; Park, Zee-Yong; Choi, Eun-Ju; Han, Weon-Cheol; Lee, Seung-Ho; Oh, Hyun-Mee; Jun, Chang-Duk

    2015-03-01

    Autoreactive T-cell responses have a crucial role in the pathology and clinical course of autoimmune diseases. Therefore, controlling the activation of these cells is an important strategy for developing therapies and therapeutics. Here, we identified that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) has a therapeutic potential for T-cell activation by modulating protein kinase C-θ (PKCθ) and its downstream pathways. Pre- and post-treatment with 4H3MC prevented IL-2 release from human transformed and untransformed T cells at the micromolar concentrations without any cytotoxic effects, in fact more efficiently than its structural analogue 4-hydroxycinnamic acid-a previously reported T-cell inhibitor. In silico analysis showed that 4H3MC is a potential inhibitor of PKC isotypes, including PKCθ-a crucial PKC isotype in T cells. Consistently, 4H3MC significantly blocked PKC activity in vitro and also inhibited the phosphorylation of PKCθ in T cells. 4H3MC had no effect on TCR-mediated membrane-proximal-signalling events such as phosphorylation of Zap70. Instead, it attenuated the phosphorylation of mitogen-activated protein kinases (ERK and p38) and promoter activities of NF-κB, AP-1 and NFAT. Taken together, our results provide the evidences that 4H3MC may have curative potential as a novel immune modulator in a broad range of immunopathological disorders by modulating PKCθ activity. PMID:25637768

  7. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  8. Activation of signalling by the activin receptor complex.

    PubMed Central

    Attisano, L; Wrana, J L; Montalvo, E; Massagué, J

    1996-01-01

    Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals. PMID:8622651

  9. Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development.

    PubMed

    Ahuja, Suchit; Dogra, Deepika; Stainier, Didier Y R; Reischauer, Sven

    2016-04-01

    The atrioventricular canal (AVC) connects the atrial and ventricular chambers of the heart and its formation is critical for the development of the cardiac valves, chamber septation and formation of the cardiac conduction system. Consequently, problems in AVC formation can lead to congenital defects ranging from cardiac arrhythmia to incomplete cardiac septation. While our knowledge about early heart tube formation is relatively comprehensive, much remains to be investigated about the genes that regulate AVC formation. Here we identify a new role for the basic helix-loop-helix factor Id4 in zebrafish AVC valve development and function. id4 is first expressed in the AVC endocardium and later becomes more highly expressed in the atrial chamber. TALEN induced inactivation of id4 causes retrograde blood flow at the AV canal under heat induced stress conditions, indicating defects in AV valve function. At the molecular level, we found that id4 inactivation causes misexpression of several genes important for AVC and AV valve formation including bmp4 and spp1. We further show that id4 appears to control the number of endocardial cells that contribute to the AV valves by regulating Wnt signaling in the developing AVC endocardium. PMID:26892463

  10. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes the vascular endothelial growth factor-stimulated MAPKs and downstream effectors AP-1 and CREB in mouse mesangial cells

    PubMed Central

    Tripathi, Satyabha; Pandey, Kailash N.

    2012-01-01

    Along with its natriuretic, diuretic, and vasodilatory properties, atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) exhibit an inhibitory effect on cell growth and proliferation. However, the signaling pathways mediating this inhibition are not well understood. The objective of this study was to determine the effect of ANP-NPRA system on mitogen-activated protein kinases (MAPKs) and the downstream proliferative transcription factors involving activating protein-1 (AP-1) and cAMP-response element binding protein (CREB) in agonist-stimulated mouse mesangial cells (MMCs). We found that ANP inhibited vascular endothelial growth factor (VEGF)-stimulated phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38), to a greater extent in NPRA-transfected cells (50–60%) relative to vector-transfected cells (25–30%). The analyses of the phosphorylated transcription factors revealed that ANP inhibited VEGF-stimulated activation of CREB, and the AP-1 subunits (c-jun and c-fos). Gel shift assays demonstrated that ANP inhibited VEGF-stimulated AP-1 and CREB DNA-binding ability by 67 % and 62 %, respectively. The addition of the protein kinase G (PKG) inhibitor, KT-5823, restored the VEGF-stimulated activation of MAPKs, AP-1, and CREB, demonstrating the integral role of cGMP/PKG signaling in NPRA-mediated effects. Our results delineate the under lying mechanisms through which ANP-NPRA system exerts an inhibitory effect on MAPKs and down-stream effector molecules, AP-1 and CREB, critical for cell growth and proliferation. PMID:22610792

  11. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

    PubMed Central

    Nezich, Catherine L.; Wang, Chunxin; Fogel, Adam I.

    2015-01-01

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats–generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy. PMID:26240184

  12. Application of activated charcoal in the downstream processing of bacterial olefinic poly(3-hydroxyalkanoates).

    PubMed

    Wampfler, Bruno; Ramsauer, Thomas; Kehl, Karl; Zinn, Manfred; Thöny-Meyer, Linda

    2010-01-01

    Medium chain length poly(hydroxyalkanoates) (mcl-PHAs) are bacterial thermoplastic elastomers with a large potential in medical applications. The present study provides a novel process to isolate and purify poly([R]-3-hydroxy-omega-undecenoate-co-3-hydroxy-omega-nonenoate-co-3-hydroxy-omega-heptenoate) (PHUE) and poly([R]-3-hydroxy-omega-undecenoate-co-3-hydroxy-omega-nonenoate-co-3-hydroxyoctanoate-co-3-hydroxy-omega-heptenoate-co-3-hydroxyhexanoate) (PHOUE) from Pseudomonas putida species. Three different types of activated charcoal were compared with regard to their capability to selectively remove impurities. The product 'Charcoal activated, powder, pure' from Merck was found to be most suitable. Using ethyl acetate as solvent, the polyesters were extracted from freeze-dried biomass at room temperature and simultaneously purified by addition of activated charcoal at the beginning of the extraction. The period of extraction was one hour and the ratio solvent to biomass was 15:1 (vol/wt). After extraction, the solids were separated by pressure filtration through a metallic lace tissue. The filtrate was again passed through the previously accumulated filter cake, followed by a second filtration through a 0.45 microm membrane to remove finest coal particles. The resulting filtrate was concentrated, thus yielding polyesters whose quality and yield depended on the quantity of activated charcoal applied. For highly pure PHUE and PHOUE with low endotoxin levels, the optimum ratio of activated charcoal to solvent for extraction (V/V) was found to be 0.5 for PHUE and 0.25 for PHOUE. The yields with regard to the raw polymers amounted to 55 wt% for PHUE and 75 wt% for PHOUE, which are acceptable for polymers that can be used for medical applications. PMID:21197841

  13. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  14. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    VEGF is one of the most critical factors that induce angiogenesis, and has thus become an attractive target for anti-angiogenesis treatment. However, most of the current anti-VEGF agents that often cause side effects cannot be recommended for long term use. Identification of natural VEGF inhibitors...

  15. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

    PubMed

    García-Rodríguez, Luis J; De Piccoli, Giacomo; Marchesi, Vanessa; Jones, Richard C; Edmondson, Ricky D; Labib, Karim

    2015-10-15

    Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint. PMID:26250113

  16. Krüppel-like Factor 6 Is a Co-activator of NF-κB That Mediates p65-dependent Transcription of Selected Downstream Genes*

    PubMed Central

    Zhang, Yu; Lei, Cao-Qi; Hu, Yun-Hong; Xia, Tian; Li, Mi; Zhong, Bo; Shu, Hong-Bing

    2014-01-01

    The transcription factor NF-κB plays a pivotal role in a broad range of physiological and pathological processes, including development, inflammation, and immunity. How NF-κB integrates activating signals to expression of specific sets of target genes is of great interest. Here, we identified Krüppel-like factor 6 (KLF6) as a co-activator of NF-κB after TNFα and IL-1β stimulation. Overexpression of KLF6 enhanced TNFα- and IL-1β-induced activation of NF-κB and transcription of a subset of downstream genes, whereas knockdown of KLF6 had opposite effects. KLF6 interacted with p65 in the nucleus and bound to the promoters of target genes. Upon IL-1β stimulation, KLF6 was recruited to promoters of a subset of NF-κB target genes in a p65-dependent manner, which was in turn required for the optimal binding of p65 to the target gene promoters. Our findings thus identified KLF6 as a previously unknown but essential co-activator of NF-κB and provided new insight into the molecular regulation of p65-dependent gene expression. PMID:24634218

  17. Biomechanical Signals Suppress TAK1 Activation to Inhibit NF-κB Transcriptional Activation in Fibrochondrocytes

    PubMed Central

    Madhavan, Shashi; Anghelina, Mirela; Sjostrom, Danen; Dossumbekova, Anar; Guttridge, Denis C.; Agarwal, Sudha

    2016-01-01

    Exercise/joint mobilization is therapeutic for inflammatory joint diseases like rheumatoid and osteoarthritis, but the mechanisms underlying its actions remain poorly understood. We report that biomechanical signals at low/physiological magnitudes are potent inhibitors of inflammation induced by diverse proinflammatory activators like IL-1β, TNF-α, and lipopolysaccharides, in fibrochondrocytes. These signals exert their anti-inflammatory effects by inhibiting phosphorylation of TAK1, a critical point where signals generated by IL-1β, TNF-α, and LPS converge to initiate NF-κB signaling cascade and proinflammatory gene induction. Additionally, biomechanical signals inhibit multiple steps in the IL-1β-induced proinflammatory cascade downstream of IκB kinase activation to regulate IκBα and IκBβ degradation and synthesis, and promote IκBα shuttling to export nuclear NF-κB and terminate its transcriptional activity. The findings demonstrate that biomechanical forces are but another important signal that uses NF-κB pathway to regulate inflammation by switching the molecular activation of discrete molecules involved in proinflammatory gene transcription. PMID:17947700

  18. NF-kB activation and its downstream target genes expression after heavy ions exposure

    NASA Astrophysics Data System (ADS)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  19. Eriocalyxin B-induced apoptosis in pancreatic adenocarcinoma cells through thiol-containing antioxidant systems and downstream signalling pathways.

    PubMed

    Li, L; Yue, G G L; Pu, J X; Sun, H D; Fung, K P; Leung, P C; Han, Q B; Lau, C B S; Leung, P S

    2014-01-01

    Thiol-containing antioxidant systems play an important role in regulating cellular redox homeostasis. Several anti-cancer agents act by targeting these systems by inducing the production of reactive oxygen species (ROS). Our earlier studies have shown that Eriocalyxin B (EriB), a diterpenoid isolated from Isodon eriocalyx, possesses anti-pancreatic tumour activities in vitro and in vivo. The present study further demonstrated that only thiol-containing antioxidants, N-acetylcysteine (NAC) or dithiothreitol (DTT), inhibited EriB-induced cytotoxicity and apoptosis. EriB suppressed the glutathione and thioredoxin antioxidant systems, thus increasing the intracellular ROS levels and regulating the MAPK, NFκB pathways. Treatment with EriB depleted the intracellular thiol-containing proteins in CAPAN-2 cells. In vivo studies also showed that EriB treatment (2.5 mg/kg) reduced the pancreatic tumour weights significantly in nude mice with increased superoxide levels. Taken together, our results shed important new light on the molecular mechanisms of EriB acting as an apoptogenic agent and its therapeutic potential for pancreatic cancer. PMID:24894173

  20. Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.

    PubMed

    Andersen, Erica F; Halloran, Mary C

    2012-10-01

    Neurons must develop complex structure to form proper connections in the nervous system. The initiation of axons in defined locations on the cell body and their extension to synaptic targets are critical steps in neuronal morphogenesis, yet the mechanisms controlling axon formation in vivo are poorly understood. The centrosome has been implicated in multiple aspects of neuronal morphogenesis; however, its function in axon development is under debate. Conflicting results from studies of centrosome function in axonogenesis suggest that its role is context dependent and underscore the importance of studying centrosome function as neurons develop in their natural environment. Using live imaging of zebrafish Rohon-Beard (RB) sensory neurons in vivo, we discovered a spatiotemporal relationship between centrosome position and the formation of RB peripheral, but not central, axons. We tested centrosome function by laser ablation and found that centrosome disruption inhibited peripheral axon outgrowth. In addition, we show that centrosome position and motility are regulated by LIM homeodomain transcription factor activity, which is specifically required for the development of RB peripheral axons. Furthermore, we show a correlation between centrosome mislocalization and ectopic axon formation in bashful (laminin alpha 1) mutants. Thus, both intrinsic transcription factor activity and extracellular cues can influence centrosome position and axon formation in vivo. This study presents the first positive association between the centrosome and axon formation in vivo and suggests that the centrosome is important for differential neurite formation in neurons with complex axonal morphologies. PMID:22899847

  1. Autocrine CSF1R signaling mediates switching between invasion and proliferation downstream of TGFβ in claudin-low breast tumor cells

    PubMed Central

    Patsialou, Antonia; Wang, Yarong; Pignatelli, Jeanine; Chen, Xiaoming; Entenberg, David; Oktay, Maja; Condeelis, John S.

    2014-01-01

    Patient data suggest that colony stimulating factor-1 (CSF1) and its receptor (CSF1R) play critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cells lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cells lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ. PMID:25088194

  2. Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling.

    PubMed

    Gerber, Simon D; Amann, Ruth; Wyder, Stefan; Trueb, Beat

    2012-01-01

    Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

  3. Comparison of the Gene Expression Profiles from Normal and Fgfrl1 Deficient Mouse Kidneys Reveals Downstream Targets of Fgfrl1 Signaling

    PubMed Central

    Gerber, Simon D.; Amann, Ruth; Wyder, Stefan; Trueb, Beat

    2012-01-01

    Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

  4. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis1[OPEN

    PubMed Central

    Kong, Qing; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-01-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  5. Androgen activates β-catenin signaling in bladder cancer cells.

    PubMed

    Li, Yi; Zheng, Yichun; Izumi, Koji; Ishiguro, Hitoshi; Ye, Bo; Li, Faqian; Miyamoto, Hiroshi

    2013-06-01

    Androgen receptor (AR) signals have been implicated in bladder carcinogenesis and tumor progression. Activation of Wnt/β-catenin signaling has also been reported to correlate with bladder cancer progression and poor patients' outcomes. However, cross talk between AR and β-catenin pathways in bladder cancer remains uncharacterized. In radical cystectomy specimens, we immunohistochemically confirmed aberrant expression of β-catenin especially in aggressive tumors. There was a strong association between nuclear expressions of AR and β-catenin in bladder tumors (P=0.0215). Kaplan-Meier and log-rank tests further revealed that reduced membranous β-catenin expression (P=0.0276), nuclear β-catenin expression (P=0.0802), and co-expression of nuclear AR and β-catenin (P=0.0043) correlated with tumor progression after cystectomy. We then assessed the effects of androgen on β-catenin in AR-positive and AR-negative bladder cancer cell lines. A synthetic androgen R1881 increased the expression of an active form of β-catenin and its downstream target c-myc only in AR-positive lines. R1881 also enhanced the activity of β-catenin-mediated transcription, which was abolished by an AR antagonist hydroxyflutamide. Using western blotting and immunofluorescence, R1881 was found to induce nuclear translocation of β-catenin when co-localized with AR. Finally, co-immunoprecipitation revealed androgen-induced associations of AR with β-catenin or T-cell factor (TCF) in bladder cancer cells. Thus, it was likely that androgen was able to activate β-catenin signaling through the AR pathway in bladder cancer cells. Our results also suggest that activation of β-catenin signaling possibly via formation of AR/β-catenin/TCF complex contributes to the progression of bladder cancer, which may enhance the feasibility of androgen deprivation as a potential therapeutic approach. PMID:23447569

  6. Cypermethrin Stimulates GSK3β-Dependent Aβ and p-tau Proteins and Cognitive Loss in Young Rats: Reduced HB-EGF Signaling and Downstream Neuroinflammation as Critical Regulators.

    PubMed

    Maurya, Shailendra Kumar; Mishra, Juhi; Abbas, Sabiya; Bandyopadhyay, Sanghamitra

    2016-03-01

    Pesticide exposure is recognized as a risk factor for Alzheimer's disease (AD). We investigated early signs of AD-like pathology upon exposure to a pyrethroid pesticide, cypermethrin, reported to impair neurodevelopment. We treated weanling rats with cypermethrin (10 and 25 mg/kg) and detected dose-dependent increase in the key proteins of AD, amyloid beta (Aβ), and phospho-tau, in frontal cortex and hippocampus as early as postnatal day 45. Upregulation of Aβ pathway involved an increase in amyloid precursor protein (APP) and its pro-amyloidogenic processing through beta-secretase (BACE) and gamma-secretase. Tau pathway entailed elevation in tau and glycogen-synthase kinase-3-beta (GSK3β)-dependent, phospho-tau. GSK3β emerged as a molecular link between the two pathways, evident from reduction in phospho-tau as well as BACE upon treating GSK3β inhibitor, lithium chloride. Exploring the mechanism revealed an attenuated heparin-binding epidermal growth factor (HB-EGF) signaling and downstream astrogliosis-mediated neuroinflammation to be responsible for inducing Aβ and phospho-tau. Cypermethrin caused a proximal reduction in HB-EGF, which promoted astrocytic nuclear factor kappa B signaling and astroglial activation close to Aβ and phospho-tau. Glial activation stimulated generation of interleukin-1 (IL-1), which upregulated GSK3β, and APP and tau as well, resulting in co-localization of Aβ and phospho-tau with IL-1 receptor. Intracerebral insertion of exogenous HB-EGF restored its own signaling and suppressed neuroinflammation and thereby Aβ and phospho-tau in cypermethrin-exposed rats, proving a central role of reduced HB-EGF signaling in cypermethrin-mediated neurodegeneration. Furthermore, cypermethrin stimulated cognitive impairments, which could be prevented by exogenous HB-EGF. Our data demonstrate that cypermethrin induces premature upregulation of GSK3β-dependent Aβ and tau pathways, where HB-EGF signaling and neuroinflammation serve as

  7. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  8. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells.

    PubMed

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    2016-08-26

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. PMID:27329811

  9. Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

    SciTech Connect

    Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

    2008-08-15

    Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.

  10. Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

    PubMed

    Wangpu, Xiongzhi; Lu, Jiaoyang; Xi, Ruxing; Yue, Fei; Sahni, Sumit; Park, Kyung Chan; Menezes, Sharleen; Huang, Michael L H; Zheng, Minhua; Kovacevic, Zaklina; Richardson, Des R

    2016-05-01

    Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway. PMID:26895766

  11. Diversity of Stability, Localization, Interaction and Control of Downstream Gene Activity in the Maize Aux/IAA Protein Family

    PubMed Central

    Ludwig, Yvonne; Berendzen, Kenneth W.; Xu, Changzheng; Piepho, Hans-Peter; Hochholdinger, Frank

    2014-01-01

    AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are central regulators of auxin signal transduction. They control many aspects of plant development, share a conserved domain structure and are localized in the nucleus. In the present study, five maize Aux/IAA proteins (ZmIAA2, ZmIAA11, ZmIAA15, ZmIAA20 and ZmIAA33) representing the evolutionary, phylogenetic and expression diversity of this gene family were characterized. Subcellular localization studies revealed that ZmIAA2, ZmIAA11 and ZmIAA15 are confined to the nucleus while ZmIAA20 and ZmIAA33 are localized in both the nucleus and the cytoplasm. Introduction of specific point mutations in the degron sequence (VGWPPV) of domain II by substituting the first proline by serine or the second proline by leucine stabilized the Aux/IAA proteins. While protein half-life times between ∼11 min (ZmIAA2) to ∼120 min (ZmIAA15) were observed in wild-type proteins, the mutated forms of all five proteins were almost as stable as GFP control proteins. Moreover, all five maize Aux/IAA proteins repressed downstream gene expression in luciferase assays to different degrees. In addition, bimolecular fluorescence complementation (BiFC) analyses demonstrated interaction of all five Aux/IAA proteins with RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1, ZmIAA10) while only ZmIAA15 and ZmIAA33 interacted with the RUM1 paralog RUL1 (RUM-LIKE 1, ZmIAA29). Moreover, ZmIAA11, ZmIAA15 ZmIAA33 displayed homotypic interaction. Hence, despite their conserved domain structure, maize Aux/IAA proteins display a significant variability in their molecular characteristics which is likely associated with the wide spectrum of their developmental functions. PMID:25203637

  12. Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

    PubMed Central

    Miao, Yi-Liang; Williams, Carmen J.

    2012-01-01

    Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

  13. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. PMID:26702834

  14. Paired inhibitory and activating receptor signals.

    PubMed

    Taylor, L S; Paul, S P; McVicar, D W

    2000-01-01

    The immunological literature has become inundated with reports regarding paired inhibitory receptors. Paired inhibitory receptor systems are highly conserved families that contain receptors involved in either cellular inhibition or activation. In most cases the paired putative biochemical antagonists are co-expressed on a given cell and thought to bind similar, if not identical, ligands making their biological role difficult to understand. Examples of these systems include immunoglobulin (Ig)-like receptors (Killer Ig Receptors, Immunoglobulin-like Transcripts/Leukocyte Ig-like Receptors/Monocyte Macrophage Ig Receptors, and Paired Ig-like Receptors), and type II lectin-like receptor systems (NKG2 and Ly49). General characteristics of these inhibitory receptors include a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). The ITIM is phosphorylated upon engagement and recruits protein tyrosine phosphatases that dephosphorylate cellular substrates that would otherwise mediate activation. In contrast, the activating receptors of these pairs use charged residues within their transmembrane domains to associate with various signal transduction chains including the gamma chain of the receptor for the Fc portion of IgE, DAP12 or DAP10. Once phosphorylated, these chains direct the signal transduction cascade resulting in cellular activation. Here we review the signaling of several paired systems and present the current models for their signal transduction cascades. PMID:11258418

  15. Curcumin exerts anti-inflammatory and antioxidative properties in 1-methyl-4-phenylpyridinium ion (MPP(+))-stimulated mesencephalic astrocytes by interference with TLR4 and downstream signaling pathway.

    PubMed

    Yu, Song; Wang, Xu; He, Xingliang; Wang, Yue; Gao, Sujie; Ren, Lu; Shi, Yan

    2016-07-01

    Neuroinflammation is closely associated with the pathophysiology of neurodegenerative diseases including Parkinson's disease (PD). Recent evidence indicates that astrocytes also play pro-inflammatory roles in the central nervous system (CNS) by activation with toll-like receptor (TLR) ligands. Therefore, targeting anti-inflammation may provide a promising therapeutic strategy for PD. Curcumin, a polyphenolic compound isolated from Curcuma longa root, has been commonly used for the treatment of neurodegenerative diseases. However, the details of how curcumin exerts neuroprotection remain uncertain. Here, we investigated the protective effect of curcumin on 1-methyl-4-phenylpyridinium ion-(MPP(+)-) stimulated primary astrocytes. Our results showed that MPP(+) stimulation resulted in significant production of tumor necrosis factor (TNF)-α, interleukin (IL-6), and reactive oxygen species (ROS) in primary mesencephalic astrocytes. Curcumin pretreatment decreased the levels of these pro-inflammatory cytokines while increased IL-10 expression in MPP(+)-stimulated astrocytes. In addition, curcumin increased the levels of antioxidant glutathione (GSH) and reduced ROS production. Our results further showed that curcumin decreased the levels of TLR4 and its downstream effectors including NF-κB, IRF3, MyD88, and TIRF that are induced by MPP(+) as well as inhibited the immunoreactivity of TLR4 and morphological activation in MPP(+)-stimulated astrocytes. Together, data suggest that curcumin might exert a beneficial effect on neuroinflammation in the pathophysiology of PD. PMID:27164829

  16. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    PubMed

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity. PMID:25670795

  17. Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells.

    PubMed

    Tanaka, Maiko; Hiragun, Takaaki; Tsutsui, Tomoko; Yanase, Yuhki; Suzuki, Hidenori; Hide, Michihiro

    2008-06-15

    Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors. PMID:18339533

  18. Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 4 (vIRF4/K10) is a novel interaction partner of CSL/CBF1, the major downstream effector of Notch signaling.

    PubMed

    Heinzelmann, Katharina; Scholz, Barbara A; Nowak, Agnes; Fossum, Even; Kremmer, Elisabeth; Haas, Juergen; Frank, Ronald; Kempkes, Bettina

    2010-12-01

    In cells infected with the Kaposi's sarcoma-associated herpesvirus (KSHV), CSL/CBF1 signaling is essential for viral replication and promotes the survival of KSHV-infected cells. CSL/CBF1 is a DNA adaptor molecule which recruits coactivator and corepressor complexes to regulate viral and cellular gene transcription and which is a major downstream effector molecule of activated Notch. The interaction of KSHV RTA and LANA with CSL/CBF1 has been shown to balance the lytic and latent viral life cycle. Here we report that a third KSHV protein, viral interferon regulatory factor 4 (vIRF4/K10), but none of the three other KSHV-encoded vIRFs, interacts with CSL/CBF1. Two regions of vIRF4 with dissimilar affinities contribute to CSL/CBF1 binding. Similar to Notch, vIRF4 targets the hydrophobic pocket in the beta trefoil domain of CSL/CBF1 through a short peptide motif which closely resembles a motif found in Notch but does not strictly follow the ΦWΦP consensus conserved in human and mouse Notch proteins. Our results suggest that vIRF4 might compete with Notch for CSL/CBF1 binding and signaling. PMID:20861242

  19. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  20. Choline-releasing glycerophosphodiesterase EDI3 links the tumor metabolome to signaling network activities.

    PubMed

    Marchan, Rosemarie; Lesjak, Michaela S; Stewart, Joanna D; Winter, Roland; Seeliger, Janine; Hengstler, Jan G

    2012-12-15

    Recently, EDI3 was identified as a key factor for choline metabolism that controls tumor cell migration and is associated with metastasis in endometrial carcinomas. EDI3 cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P). Choline is then further metabolized to phosphatidylcholine (PtdC), the major lipid in membranes and a key player in membrane-mediated cell signaling. The second product, G3P, is a precursor molecule for several lipids with central roles in signaling, for example lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG). LPA activates intracellular signaling pathways by binding to specific LPA receptors, including membrane-bound G protein-coupled receptors and the intracellular nuclear receptor, PPARγ. Conversely, PA and DAG mediate signaling by acting as lipid anchors that bind and activate several signaling proteins. For example, binding of GTPases and PKC to PA and DAG, respectively, increases the activation of signaling networks, mediating processes such as migration, adhesion, proliferation or anti-apoptosis-all relevant for tumor development. We present a concept by which EDI3 either directly generates signaling molecules or provides "membrane anchors" for downstream signaling factors. As a result, EDI3 links choline metabolism to signaling activities resulting in a more malignant phenotype. PMID:23114620

  1. Characterization of the mouse beta maj globin transcription termination region: a spacing sequence is required between the poly(A) signal sequence and multiple downstream termination elements.

    PubMed Central

    Tantravahi, J; Alvira, M; Falck-Pedersen, E

    1993-01-01

    For the majority of mRNA encoding eukaryotic transcription units, there is little or no knowledge of the elements responsible for transcription termination or how they may interact with RNA polymerase. In this report, we have used recombinant adenovirus reporter vectors to characterize the mouse beta maj globin sequence elements that cause transcription termination. Within the globin 3' termination region, we have identified at least three sequence elements which induce significant levels of transcription termination (> 50%). The smallest functionally active element (64% termination) is 69 bp in length. The natural arrangement of these elements results in a cumulative termination which is greater than 90%. Recognition of the termination elements by RNA polymerase II depends on the presence of a functional poly(A) signal sequence. We demonstrate that efficient transcription termination depends on appropriate spacing between the poly(A) signal sequence and the termination element. Images PMID:8417354

  2. Pharmacological assessments of nitric oxide synthase isoforms and downstream diversity of NO signaling in the maintenance of thermal and mechanical hypersensitivity after peripheral nerve injury in mice.

    PubMed

    Tanabe, Mitsuo; Nagatani, Yoshinori; Saitoh, Kazuya; Takasu, Keiko; Ono, Hideki

    2009-03-01

    Nitric oxide synthase (NOS) isoforms and NO downstream signal pathways involved spinally in the maintenance of thermal and mechanical hypersensitivity were assessed in a mouse model of neuropathic pain developing after partial ligation of the sciatic nerve. Intrathecal injection of the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), the highly selective neuronal NOS (nNOS) inhibitor N(omega)-propyl-l-arginine and the potent selective inducible NOS (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride (AMT) exerted dose-dependent analgesic effects on thermal and mechanical hypersensitivity, which were assessed by the plantar and von Frey tests, respectively, suggesting that both nNOS and iNOS participate in producing NO to maintain neuropathic pain. Since the selective inhibitor of NO-sensitive guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor Rp-8-pCPT-cGMPS intrathecally exerted dose-dependent analgesic effects on thermal and mechanical hypersensitivity, spinally released NO most likely stimulates the NO-cGMP-PKG pathway. Moreover, the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), a potent superoxide scavenger, reduced thermal and mechanical hypersensitivity when administered intrathecally, suggesting that spinal release of superoxide, which can then react with NO to produce peroxynitrite, also appears to mediate neuropathic pain. Finally, intrathecal injection of phenyl-N-tert-butylnitrone (PBN), a reactive oxygen species (ROS) scavenger, ameliorated thermal and mechanical hypersensitivity, thus further confirming the importance of ROS including NO and superoxide in the maintenance of neuropathic pain. Together, the present results demonstrate that NO, produced presumably via nNOS and iNOS in the spinal cord, mediates the maintenance of neuropathic pain following peripheral nerve

  3. Sp and GATA factors are critical for Apolipoprotein AI downstream enhancer activity in human HepG2 cells.

    PubMed

    Ivanov, Gleb S; Kater, Jessie M; Jha, Shivkumar H; Stutius, Erica A; Sabharwal, Ravleen; Tricarico, Marisa D; Ginsburg, Geoffrey S; Ozer, Josef S

    2003-12-24

    The factors that bind to the hepatic-specific human apolipoprotein AI (apoAI) 48-bp downstream enhancer (DSE) were identified and characterized by electrophoretic mobility shift assays. A significant homology was shown between the histone 4 (H4) promoters and the hepatic-specific human apoAI DSE at Sp1 and H4TF2 binding sites. Human HepG2 nuclear extracts were used to form four specific complexes with the DSE (referred to as apoAI DSE-1, -2, -3, and -4). The apoAI DSE-1 and -2 complexes showed similar binding specificity to the Sp/H4TF1 consensus site within the apoAI DSE. The apoAI DSE-1 complex was predominantly recognized by anti-Sp1 and Sp3 sera in gel shift assays, indicating that the DSE was recognized by multiple Sp family members. Nuclear extracts that were prepared from retinoic acid treated HepG2 cells showed increased levels of Sp factors in gel shift and Western blot assays. The apoAI DSE-2 complex was identified as H4TF1 and formed in the absence of magnesium chloride. The apoAI DSE-3 complex bound to a consensus GATA element within the DSE that was recognized by recombinant human GATA-6 as well. The apoAI DSE-3 complex was completely disrupted by a GATA-4 antibody in EMSA. GATA-4 and -6 were detected in nuclear extracts prepared from retinoic acid treated HepG2 cells using Western blot assays. The highest apoAI DSE-3 levels were observed with retinoic acid treated HepG2 cell nuclear extracts in EMSA. ApoAI DSE-4 is a multi-factor complex that includes an Sp/H4TF1 factor and either H4TF2 or apoAI DSE-3. Because apoAI DSE mutations revealed transcription defects in transient transfection assays, we conclude that the entire DSE sequence is required for full apoAI transcriptional activity in HepG2 cells. PMID:14659877

  4. Transcript Expression Data from Human Islets Links Regulatory Signals from Genome-Wide Association Studies for Type 2 Diabetes and Glycemic Traits to Their Downstream Effectors.

    PubMed

    van de Bunt, Martijn; Manning Fox, Jocelyn E; Dai, Xiaoqing; Barrett, Amy; Grey, Caleb; Li, Lei; Bennett, Amanda J; Johnson, Paul R; Rajotte, Raymond V; Gaulton, Kyle J; Dermitzakis, Emmanouil T; MacDonald, Patrick E; McCarthy, Mark I; Gloyn, Anna L

    2015-12-01

    The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. ‎At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci. PMID:26624892

  5. Identification of Phosphotyrosine Binding Domain-Containing Proteins as Novel Downstream Targets of the EphA8 Signaling Function▿ †

    PubMed Central

    Shin, Jongdae; Gu, Changkyu; Park, Eunjeong; Park, Soochul

    2007-01-01

    Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway. PMID:17875921

  6. Transcript Expression Data from Human Islets Links Regulatory Signals from Genome-Wide Association Studies for Type 2 Diabetes and Glycemic Traits to Their Downstream Effectors

    PubMed Central

    Dai, Xiaoqing; Barrett, Amy; Grey, Caleb; Li, Lei; Bennett, Amanda J.; Johnson, Paul R.; Rajotte, Raymond V.; Gaulton, Kyle J.; Dermitzakis, Emmanouil T.; MacDonald, Patrick E.; McCarthy, Mark I.; Gloyn, Anna L.

    2015-01-01

    The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. ‎At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci. PMID:26624892

  7. Notum deacylates Wnts to suppress signalling activity

    PubMed Central

    Howell, Steve; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O’Reilly, Nicola; Snijders, Ambrosius P.; Jones, E. Yvonne; Vincent, Jean-Paul

    2015-01-01

    Signalling by Wnts is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnts from the cell surface. However, this view fails to explain specificity since glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which likely help Notum colocalise with Wnts. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnts and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  8. Up-stream and Down-stream Events in the NF-kB Activation Cascade in Response to Sparsely and Densely Ionizing Radiation

    NASA Astrophysics Data System (ADS)

    Langen, Britta; Hellweg, Christine; Baumstark-Khan, Christa; Ruscher, Roland; Schmitz, Claudia; Arenz, Andrea; Lau, Patrick; Meier, Matthias M.; Testard, Isabelle; Reitz, Guenther

    Radiation is a potentially limiting factor for long term orbital and interplanetary missions. Long-term exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. As an antiapoptotic factor, if activated in human cells by exposure to components of cosmic rays, the transcription factor nuclear factor κB (NF- κB) could influence the cancer risk of astronauts exposed to cosmic radiation and improve cellular survival after exposure to high radiation doses. In previous studies using a screening assay for the detection of NF-κB-dependent gene induction (HEK-pNF-κB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown (Radiat. Res. 164: 527- 530, 2005). In this work, the events upstream of NF-κB attachment to its promoter and enhancer binding sites, and downstream expression of target genes were analysed. It is supposed that the ATM kinase mediates the signal from damaged DNA in the nucleus to kinases in the cytoplasm, such as NF-κB essential modulator (NEMO). For liberation of NF-κB and its nuclear translocation, the inhibitor of NF-κB (IκB) has to be degraded in the proteasom. In order to evaluate a role of NEMO in the radiation response, the survival of murine embryonic fibroblasts expressing wildtype NEMO and lacking NEMO in response to ionizing radiation was analyzed. Lack of NEMO impairs survival after X-ray exposure. The inhibition of ATM by KU-55933 suppresses the X-ray and heavy ion (13C, 35 MeV/u, LET 70 keV/µm) induced activation of NF-κB dependent gene expression, indicating the central position of ATM in radiation induced NF-κB activation. Short-term incubation with the proteasome inhibitor MG-132 also blocks NF-κB activation by radiation. These results suggest a role of

  9. Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ).

    PubMed

    Gandhirajan, Rajesh Kumar; Jain, Manaswita; Walla, Benedikt; Johnsen, Marc; Bartram, Malte P; Huynh Anh, Minh; Rinschen, Markus M; Benzing, Thomas; Schermer, Bernhard

    2016-05-27

    Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway. PMID:27048650

  10. The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Xiong, Lingxin; Xie, Jingshu; Song, Chenxue; Liu, Jinping; Zheng, Jingtong; Liu, Chuangui; Zhang, Xiaotian; Li, Pingya; Wang, Fang

    2015-01-01

    Epidemiological studies have verified the critical role that antioxidative stress plays in protecting vascular endothelial cells. The aims of the present study were to investigate the antioxidative activities and differential regulation of nuclear erythroid-related factor 2- (Nrf2-) mediated gene expression by Xueshuan Xinmaining Tablet (XXT), a traditional Chinese medicine with the effect of treating cardiovascular diseases. The antioxidative activities of XXT were investigated using quantitative real-time PCR (qPCR), a PCR array, and western blotting. Our results indicated that XXT exhibited potent antioxidative activities by suppressing the levels of hydrogen peroxide- (H2O2-) induced reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs). We were also conscious of strong Nrf2-mediated antioxidant induction. XXT enhanced the expressions of Keap1, Nrf2, and Nrf2-mediated genes, such as glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H: quinine oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), and glutathione peroxidase (GPX) in HUVECs. In summary, XXT strongly activated Nrf2 and its downstream regulated genes, which may contribute to the antioxidative and vascular endothelial cell protective activities of XXT. PMID:26681964

  11. Characterising low molecular weight dissolved organic carbon compounds in subglacial systems; implications for subglacial metabolic activity and potential downstream export

    NASA Astrophysics Data System (ADS)

    Lawson, Emily; Wadham, Jemma; Lis, Grzegorz; Telling, Jon

    2010-05-01

    Glaciers and ice sheets represent ~10% of the contemporary global surface coverage, yet remain one of the least explored sectors of the Earth's biosphere. The basal regions of these ice masses, known as subglacial environments, are capable of harbouring a diverse range of microorganisms that are often metabolically active despite the lack of sunlight, the cold temperatures and nutrient scarcity. Here, we consider the potential for such environments to be active components of the Earth's biogeochemical cycles. Subglacial environments have traditionally been excluded from global carbon budgets because they were assumed to be predominantly abiotic. Organic carbon (OC) reservoirs and transformations were also believed to be limited. However, significant stores of bioavailable carbon are thought to be present in glacially-overridden material, providing a potential substrate for in situ microbial metabolism. We examine the molecular characteristics of dissolved OC in basal ice and subglacial runoff from two glacier/ice-sheet systems with contrasting organic carbon substrates; Russell/Leverett Glacier, Greenland ice sheet, and Engabreen, Norway, to determine the range of dissolved low molecular weight OC (LMWOC) compounds and their relative bioavailability. Overridden material beneath the Greenland ice sheet is relatively young and organic-rich, contrasting with the older crystalline bedrock/continental shield that was overridden during glaciation at Engabreen. We first utilise a combination of fluorescence spectroscopy and ion chromatography to identify and quantify volatile fatty acids, carbohydrates and amino acids in basal ice. Volatile fatty acids are key metabolic substrates and their provision is thought to be a primary control on subglacial metabolic activity. We then provide a temporal record of amino acids and carbohydrates in subglacial runoff from Leverett Glacier (June 23rd - August 18th 2009), and compare this with subglacial runoff from Engabreen (2008 melt

  12. Analysis of the effects of human activities on the hydromorphological evolution channel of the Saint-Maurice River downstream from La Gabelle dam (Quebec, Canada)

    NASA Astrophysics Data System (ADS)

    Vadnais, Marie-Ève; Assani, Ali A.; Landry, Raphaëlle; Leroux, Denis; Gratton, Denis

    2012-11-01

    During the first half of the twentieth century, many hydroelectric facilities were built in the Saint-Maurice River watershed, followed by other human activities in the second half of the century (pleasure boating, boom dismantling, urbanization, etc.). The goal of the study is to constrain the effects of these various types of human activities, particularly those of the many dams in the watershed, on the hydromorphological evolution of the Saint-Maurice River downstream from the La Gabelle (dam) power plant (43,000 km2). Comparison of specific discharge in this river with streamflow measured in a natural river setting reveals a significant decrease in seasonal maximum flows, aside from winter, when daily maximum flows increased significantly. Also, unlike natural rivers, the long-term trend in spring flows is not characterized by a significant change in mean downstream from the La Gabelle plant. These hydrological changes are linked to the inversion-type management mode of the reservoirs built downstream from the plant. As for the morphological evolution, the longitudinal variability of bankfull width downstream from the plant shows two significant shifts in mean: the first, which was quasi-abrupt, took place downstream of the des Forges rapid; and the second, which was gradual, occurred upstream from the confluence of the Saint-Maurice River with the St. Lawrence River, above the point where the Saint-Maurice splits into two branches. Comparison of aerial photographs taken at various times (1948, 1964, 1975, 1996, and 2008) reveals no significant change in the mean of bankfull width over time. However, a significant increase in the surface area of islets located at the confluence was observed, which is caused by sediment accumulation. These sediments were likely derived from local bank erosion resulting from anthropogenic changes.

  13. An Upstream By-product from Ester Activation via NHC-Catalysis Catalyzes Downstream Sulfonyl Migration Reaction.

    PubMed

    Han, Runfeng; He, Liwenze; Liu, Lin; Xie, Xingang; She, Xuegong

    2016-01-01

    A sequential reaction combining N-heterocyclic carbene (NHC) and N-hydroxyphthalimide (NHPI) catalysis allowed for the upstream by-product NHPI, which was generated in the NHC-catalyzed cycloaddition reaction, to act as the catalyst for a downstream nitrogen-to-carbon sulfonyl migration reaction. Enantiomeric excess of the major product in the cycloaddition reaction remained intact in the follow-up sulfonyl migration reaction. PMID:26522328

  14. Aurora A drives early signalling and vesicle dynamics during T-cell activation

    PubMed Central

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B.; Sánchez-Madrid, Francisco

    2016-01-01

    Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

  15. Aurora A drives early signalling and vesicle dynamics during T-cell activation.

    PubMed

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B; Sánchez-Madrid, Francisco

    2016-01-01

    Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

  16. The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T- cell acute lymphoblastic leukemia: A report from the Children's Oncology Group

    PubMed Central

    Gedman, Amanda Larson; Chen, Qing; Desmoulin, Sita Kugel; Ge, Yubin; LaFiura, Katherine; Haska, Christina L.; Cherian, Christina; Devidas, Meenakshi; Linda, Stephen B.; Taub, Jeffrey W.; Matherly, Larry H.

    2009-01-01

    We explored the impact of mutations in the NOTCH1, FBW7 and PTEN genes on prognosis and downstream signaling in a well-defined cohort of 47 pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. In T-ALL lymphoblasts, we identified high frequency mutations in NOTCH1 (n=16), FBW7 (n=5) and PTEN (n=26). NOTCH1 mutations resulted in 1.3-3.3-fold increased transactivation of a HES1 reporter construct over wild-type NOTCH1; mutant FBW7 resulted in further augmentation of reporter gene activity. NOTCH1 and FBW7 mutations were accompanied by increased median transcripts for NOTCH1 target genes (HES1, DELTEX1, cMYC). However, none of these mutations were associated with treatment outcome. Elevated HES1, DELTEX1 and cMYC transcripts were associated with significant increases in transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltranserase, Bcl-2 and dihydrofolate reductase. PTEN transcripts positively correlated with HES1 and cMYC transcript levels. Our results suggest that (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL, and (2) depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted. PMID:19340001

  17. MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells.

    PubMed

    Das, Falguni; Dey, Nirmalya; Bera, Amit; Kasinath, Balakuntalam S; Ghosh-Choudhury, Nandini; Choudhury, Goutam Ghosh

    2016-07-01

    Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1. PMID:27226530

  18. Sleep Loss Activates Cellular Inflammatory Signaling

    PubMed Central

    Irwin, Michael R.; Wang, Minge; Ribeiro, Denise; Cho, Hyong Jin; Olmstead, Richard; Breen, Elizabeth Crabb; Martinez-Maza, Otoniel; Cole, Steve

    2008-01-01

    Background Accumulating evidence suggests that sleep disturbance is associated with inflammation and related disorders including cardiovascular disease, arthritis, and diabetes mellitus. This study was undertaken to test the effects of sleep loss on activation of nuclear factor (NF) -κB, a transcription factor that serves a critical role in the inflammatory signaling cascade. Methods In 14 healthy adults (7 females; 7 males), peripheral blood mononuclear cell NF-κB was repeatedly assessed, along with enumeration of lymphocyte subpopulations, in the morning after baseline sleep, partial sleep deprivation (awake from 23:00 h to 03:00 h), and recovery sleep. Results In the morning after a night of sleep loss, mononuclear cell NF-κB activation was significantly greater compared with morning levels following uninterrupted baseline or recovery sleep, in which the response was found in females but not in males. Conclusions These results identify NF-κB activation as a molecular pathway by which sleep disturbance may influence leukocyte inflammatory gene expression and the risk of inflammation-related disease. PMID:18561896

  19. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  20. Heat dissipation guides activation in signaling proteins.

    PubMed

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  1. Asymmetric activation of Dll4-Notch signaling by Foxn4 and proneural factors activates BMP/TGFβ signaling to specify V2b interneurons in the spinal cord

    PubMed Central

    Misra, Kamana; Luo, Huijun; Li, Shengguo; Matise, Michael; Xiang, Mengqing

    2014-01-01

    During development of the ventral spinal cord, the V2 interneurons emerge from p2 progenitors and diversify into two major subtypes, V2a and V2b, that play key roles in locomotor coordination. Dll4-mediated Notch activation in a subset of p2 precursors constitutes the crucial first step towards generating neuronal diversity in this domain. The mechanism behind the asymmetric Notch activation and downstream signaling events are, however, unknown at present. We show here that the Ascl1 and Neurog basic helix-loop-helix (bHLH) proneural factors are expressed in a mosaic pattern in p2 progenitors and that Foxn4 is required for setting and maintaining this expression mosaic. By binding directly to a conserved Dll4 enhancer, Foxn4 and Ascl1 activate Dll4 expression, whereas Neurog proteins prevent this effect, thereby resulting in asymmetric activation of Dll4 expression in V2 precursors expressing different combinations of proneural and Foxn4 transcription factors. Lineage tracing using the Cre-LoxP system reveals selective expression of Dll4 in V2a precursors, whereas Dll4 expression is initially excluded from V2b precursors. We provide evidence that BMP/TGFβ signaling is activated in V2b precursors and that Dll4-mediated Notch signaling is responsible for this activation. Using a gain-of-function approach and by inhibiting BMP/TGFβ signal transduction with pathway antagonists and RNAi knockdown, we further demonstrate that BMP/TGFβ signaling is both necessary and sufficient for V2b fate specification. Our data together thus suggest that the mosaic expression of Foxn4 and proneural factors may serve as the trigger to initiate asymmetric Dll4-Notch and subsequent BMP/TGFβ signaling events required for neuronal diversity in the V2 domain. PMID:24257627

  2. Active voltammetric microsensors with neural signal processing.

    SciTech Connect

    Vogt, M. C.

    1998-12-11

    Many industrial and environmental processes, including bioremediation, would benefit from the feedback and control information provided by a local multi-analyte chemical sensor. For most processes, such a sensor would need to be rugged enough to be placed in situ for long-term remote monitoring, and inexpensive enough to be fielded in useful numbers. The multi-analyte capability is difficult to obtain from common passive sensors, but can be provided by an active device that produces a spectrum-type response. Such new active gas microsensor technology has been developed at Argonne National Laboratory. The technology couples an electrocatalytic ceramic-metallic (cermet) microsensor with a voltammetric measurement technique and advanced neural signal processing. It has been demonstrated to be flexible, rugged, and very economical to produce and deploy. Both narrow interest detectors and wide spectrum instruments have been developed around this technology. Much of this technology's strength lies in the active measurement technique employed. The technique involves applying voltammetry to a miniature electrocatalytic cell to produce unique chemical ''signatures'' from the analytes. These signatures are processed with neural pattern recognition algorithms to identify and quantify the components in the analyte. The neural signal processing allows for innovative sampling and analysis strategies to be employed with the microsensor. In most situations, the whole response signature from the voltammogram can be used to identify, classify, and quantify an analyte, without dissecting it into component parts. This allows an instrument to be calibrated once for a specific gas or mixture of gases by simple exposure to a multi-component standard rather than by a series of individual gases. The sampled unknown analytes can vary in composition or in concentration, the calibration, sensing, and processing methods of these active voltammetric microsensors can detect, recognize, and

  3. Activation of Akt Signaling in Prostate Induces a TGFβ Mediated Restraint on Cancer Progression and Metastasis

    PubMed Central

    Bjerke, Glen A.; Yang, Chun-Song; Frierson, Henry F.; Paschal, Bryce M.; Wotton, David

    2014-01-01

    Mutations in the PTEN tumor suppressor gene are found in a high proportion of human prostate cancers, and in mice, Pten deletion induces high-grade prostate intra-epithelial neoplasia (HGPIN). However, progression from HGPIN to invasive cancer occurs slowly, suggesting that tumorigenesis is subject to restraint. We show that Pten deletion, or constitutive activation of the downstream kinase AKT, activates the transforming growth factor (TGF) β pathway in prostate epithelial cells. TGFβ signaling is known to play a tumor suppressive role in many cancer types, and reduced expression of TGFβ receptors correlates with advanced human prostate cancer. We demonstrate that in combination either with loss of Pten, or expression of constitutively active AKT1, inactivation of TGFβ signaling by deletion of the TGFβ type II receptor gene relieves a restraint on tumorigenesis. This results in rapid progession to lethal prostate cancer, including metastasis to lymph node and lung. In prostate epithelium, inactivation of TGFβ signaling alone is insufficient to initiate tumorigenesis, but greatly accelerates cancer progression. The activation of TGFβ signaling by Pten loss or AKT activation suggests that the same signaling events that play key roles in tumor initiation also induce the activity of a pathway that restrains disease progression. PMID:23995785

  4. Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling

    PubMed Central

    Gami, Minaxi S; Iser, Wendy B; Hanselman, Keaton B; Wolkow, Catherine A

    2006-01-01

    Background In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals. Results This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes. Conclusion A

  5. Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light

    NASA Astrophysics Data System (ADS)

    Lin, Liang; Liu, Ling; Zhao, Bing; Xie, Ran; Lin, Wei; Li, He; Li, Yaya; Shi, Minlong; Chen, Ye-Guang; Springer, Timothy A.; Chen, Xing

    2015-05-01

    Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor β (TGF-β) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon near-infrared irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β signalling in living mice.

  6. Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light.

    PubMed

    Lin, Liang; Liu, Ling; Zhao, Bing; Xie, Ran; Lin, Wei; Li, He; Li, Yaya; Shi, Minlong; Chen, Ye-Guang; Springer, Timothy A; Chen, Xing

    2015-05-01

    Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor β (TGF-β) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon near-infrared irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β signalling in living mice. PMID:25775150

  7. Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

    PubMed Central

    Cahill, Emma; Salery, Marine; Vanhoutte, Peter; Caboche, Jocelyne

    2014-01-01

    Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse. PMID:24409148

  8. PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling.

    PubMed

    Xue, Kai; Song, Jiazhe; Yang, Yan; Li, Zhi; Wu, Chunhua; Jin, Jinhua; Li, Wenzhe

    2016-05-01

    PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells. PMID:27016671

  9. Targeting pathways downstream of KRAS in lung adenocarcinoma.

    PubMed

    Zhu, Zehua; Golay, Hadrien G; Barbie, David A

    2014-08-01

    Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure. PMID:25303301

  10. Targeting pathways downstream of KRAS in lung adenocarcinoma

    PubMed Central

    Zhu, Zehua; Golay, Hadrien G; Barbie, David A

    2014-01-01

    Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure. PMID:25303301

  11. Notch Activity Modulates the Responsiveness of Neural Progenitors to Sonic Hedgehog Signaling

    PubMed Central

    Kong, Jennifer H.; Yang, Linlin; Dessaud, Eric; Chuang, Katherine; Moore, Destaye M.; Rohatgi, Rajat; Briscoe, James; Novitch, Bennett G.

    2015-01-01

    Summary Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination. PMID:25936505

  12. Notch1 Signaling Is Activated in Cells Expressing Embryonic Stem Cell Proteins in Human Primary Nasopharyngeal Carcinoma

    PubMed Central

    Zhang, Yue; Peng, Jianhua; Zhang, Huxiang; Zhu, Yi; Wan, Li; Chen, Jianfu; Chen, Xiaoyun; Lin, Renyu; Li, He; Mao, XiaoOu; Jin, Kunlin

    2010-01-01

    Objective: To explore the expression of Notch1 signaling pathway in nasopharyngeal carcinoma (NPC). Methods: We performed immunocytochemistry on surgically resected NPC using antibodies against embryonic stem (ES) cell proteins and against Notch1 signaling components. Results: We found that ES cell protein markers SOX2 and OCT4 were expressed in a subpopulation of cells for all three subtypes of NPC but barely in the normal control. Double immunostaining shows that SOX2- and OCT4-positive cells coexpressed proliferative markers, suggesting that human NPC may contain cancer stem–like cells. In addition, we found that Notch1 signaling was activated in NPC. Confocal images show that the Notch1 signaling activated form and Hes1, a downstream target of Notch1 signaling, was predominantly found in SOX2- and OCT4-positive cells. Conclusion: Our findings suggest that the Notch1 signaling pathway might be a regulator of cancer stem–like cells in NPC. PMID:20211102

  13. New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity.

    PubMed

    Jeong, A-Reum; Lee, Si-Seok; Han, Yun-Jeong; Shin, Ah-Young; Baek, Ayoung; Ahn, Taeho; Kim, Min-Gon; Kim, Young Soon; Lee, Keun Woo; Nagatani, Akira; Kim, Jeong-Il

    2016-08-01

    Plant phytochromes are photoreceptors that mediate a variety of photomorphogenic responses. There are two spectral photoisomers, the red light-absorbing Pr and far-red light-absorbing Pfr forms, and the photoreversible transformation between the two forms is important for the functioning of phytochromes. In this study, we isolated a Tyr-268-to-Val mutant of Avena sativa phytochrome A (AsYVA) that displayed little photoconversion. Interestingly, transgenic plants of AsYVA showed light-independent phytochrome signaling with a constitutive photomorphogenic (cop) phenotype that is characterized by shortened hypocotyls and open cotyledons in the dark. In addition, the corresponding Tyr-303-to-Val mutant of Arabidopsis (Arabidopsis thaliana) phytochrome B (AtYVB) exhibited nuclear localization and interaction with phytochrome-interacting factor 3 (PIF3) independently of light, conferring a constitutive photomorphogenic development to its transgenic plants, which is comparable to the first constitutively active version of phytochrome B (YHB; Tyr-276-to-His mutant). We also found that chromophore ligation was required for the light-independent interaction of AtYVB with PIF3. Moreover, we demonstrated that AtYVB did not exhibit phytochrome B activity when it was localized in the cytosol by fusion with the nuclear export signal and that AsYVA exhibited the full activity of phytochrome A when localized in the nucleus by fusion with the nuclear localization signal. Furthermore, the corresponding Tyr-269-to-Val mutant of Arabidopsis phytochrome A (AtYVA) exhibited similar cop phenotypes in transgenic plants to AsYVA. Collectively, these results suggest that the conserved Tyr residues in the chromophore-binding pocket play an important role during the Pr-to-Pfr photoconversion of phytochromes, providing new constitutively active alleles of phytochromes by the Tyr-to-Val mutation. PMID:27325667

  14. K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

    PubMed Central

    Katsnelson, Michael A.; Rucker, L. Graham; Russo, Hana M.; Dubyak, George R.

    2015-01-01

    Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal. PMID:25762778

  15. IL-1 signaling modulates STAT activation to antagonize retinoic acid signaling and control Th17–iTreg balance

    PubMed Central

    Basu, Rajatava; Whitley, Sarah K.; Bhaumik, Suniti; Zindl, Carlene L.; Schoeb, Trenton R.; Benveniste, Etty N.; Pear, Warren S.; Hatton, Robin D.; Weaver, Casey T.

    2016-01-01

    Interleukin 17 (IL-17)-producing helper (TH17) and inducible regulatory CD4+ T (iTreg) cells emerge from an overlapping developmental program. In the intestines, the vitamin A metabolite retinoic acid (RA) is produced at steady state and acts as an important cofactor to induce iTreg cell development while potently inhibiting TH17 development. Here, we found that IL-1 was required to fully override RA-mediated Foxp3 expression and induce protective TH17 responses. Through induction of an NF-κB-dependent repression of SOCS3 expression, IL-1 increased the amplitude and duration of STAT3 phosphorylation induced by TH17-polarizing cytokines, leading to an altered balance of STAT3–STAT5 binding to shared consensus sequences in developing T cells. Thus, IL-1 signaling differentially modulated STAT activation downstream of cytokine receptors to control TH17–iTreg developmental fate. PMID:25642823

  16. p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation.

    PubMed

    Sohn, Dennis; Essmann, Frank; Schulze-Osthoff, Klaus; Jänicke, Reiner U

    2006-12-01

    The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria. PMID:17145870

  17. Adenomatous polyposis coli (APC) regulates multiple signaling pathways by enhancing glycogen synthase kinase-3 (GSK-3) activity.

    PubMed

    Valvezan, Alexander J; Zhang, Fang; Diehl, J Alan; Klein, Peter S

    2012-02-01

    Glycogen synthase kinase-3 (GSK-3) is essential for many signaling pathways and cellular processes. As Adenomatous Polyposis Coli (APC) functions in many of the same processes, we investigated a role for APC in the regulation of GSK-3-dependent signaling. We find that APC directly enhances GSK-3 activity. Furthermore, knockdown of APC mimics inhibition of GSK-3 by reducing phosphorylation of glycogen synthase and by activating mTOR, revealing novel roles for APC in the regulation of these enzymes. Wnt signaling inhibits GSK-3 through an unknown mechanism, and this results in both stabilization of β-catenin and activation of mTOR. We therefore hypothesized that Wnts may regulate GSK-3 by disrupting the interaction between APC and the Axin-GSK-3 complex. We find that Wnts rapidly induce APC dissociation from Axin, correlating with β-catenin stabilization. Furthermore, Axin interaction with the Wnt co-receptor LRP6 causes APC dissociation from Axin. We propose that APC regulates multiple signaling pathways by enhancing GSK-3 activity, and that Wnts induce APC dissociation from Axin to reduce GSK-3 activity and activate downstream signaling. APC regulation of GSK-3 also provides a novel mechanism for Wnt regulation of multiple downstream effectors, including β-catenin and mTOR. PMID:22184111

  18. Ras-activated Dsor1 promotes Wnt signaling in Drosophila development.

    PubMed

    Hall, Eric T; Verheyen, Esther M

    2015-12-15

    Wnt/Wingless (Wg) and Ras-MAPK signaling both play fundamental roles in growth and cell fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras-MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. We find novel evidence that the Drosophila homolog of the dual specificity kinases MEK1/2 (also known as MAP2K1/2), Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). We identify a close physical interaction between Dsor1 and Arm, and find that catalytically inactive Dsor1 causes a reduction in active Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. We find that Ras-Dsor1 activity is independent of upstream activation by EGFR, and instead it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Taken together, our results suggest that there is a new crosstalk pathway between insulin and Wg signaling that is mediated by Dsor1. PMID:26542023

  19. Ionizing Radiation Shifts the PAI-1/ID-1 Balance and Activates Notch Signaling in Endothelial Cells

    SciTech Connect

    Scharpfenecker, Marion; Kruse, Jacqueline; Sprong, Debbie; Russell, Nicola S.; Dijke, Peter ten; Stewart, Fiona A.

    2009-02-01

    Purpose: Transforming growth factor-{beta} (TGF-{beta}) and Notch signaling pathways are important regulators of vascular homeostasis and vessel remodeling; mutations in these pathways can lead to vascular disorders. Similar vascular phenotypes develop in the normal tissues of cancer patients as a long-term effect of radiotherapy. Irradiation most severely affects the capillaries, which become leaky and dilated and might eventually rupture. To investigate the mechanism of such capillary damage, we studied the effect of TGF-{beta} and Notch signaling in microvascular endothelial cells. Methods and Materials: Human microvascular endothelial cells were irradiated with 5 or 10 Gy and activation of TGF-{beta} and Notch signaling pathways was assessed by biochemical methods and a cell migration assay. Results: Ionizing radiation induced Smad2 phosphorylation and nuclear translocation and increased mRNA and protein expression of the activin-like kinase 5 (ALK5) target gene plasminogen activator inhibitor-1 (PAI-1). At the same time, we observed diminished Smad1/5/8 activation and downregulation of the ALK1 downstream target, inhibitor of DNA binding-1 (ID-1). We also measured an upregulation of the Notch ligand Jagged-1 and the target gene Hey1. Decreased inhibitor of DNA binding-1 levels coincided with a reduced ability of the cells to migrate. Conclusion: Ionizing radiation shifts the balance from ALK1 to ALK5 signaling and activates the Notch pathway in endothelial cells. This combination of anti-angiogenic signals contributes to reduced cell migration after irradiation.

  20. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    NASA Astrophysics Data System (ADS)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  1. Ultraviolet radiation-induced inflammation activates β-catenin signaling in mouse skin and skin tumors.

    PubMed

    Prasad, Ram; Katiyar, Santosh K

    2014-04-01

    UVB-induced inflammation, in particular the overexpression of cyclooxygenase-2 (COX-2) and prostaglandin (PG) E2, has been implicated in photocarcinogenesis. UVB-induced COX-2 has been associated with β-catenin signaling in keratinocytes. However, a definitive role for COX-2 in the activation of β-catenin signaling as well as its role in UVB-induced skin tumors has not been established. We report that exposure of the skin to UVB resulted in a time- and dose-dependent activation of β-catenin in C3H/HeN mice. This response was COX-2-dependent as UVB-exposed COX-2-deficient mice exhibited significantly lower levels of UVB-induced activation of β-catenin. Moreover, treatment of mice with indomethacin, a COX-2 inhibitor, and an EP2 antagonist inhibited UVB-induced β-catenin signaling. Exposure of SKH-1 hairless mice to UVB radiation (180 mJ/cm2) 3 times a week for 24 weeks resulted in activation of β-catenin signaling in UVB-irradiated skin as well as UVB-induced skin tumors. Concomitantly, the levels of CK1α and GSK-3β, which are responsible for β-catenin signaling, were reduced while the levels of c-Myc and cyclin D1, which are downstream targets of β-catenin, were increased. To further verify the role of UVB-induced inflammation in activation of β-catenin signaling, a high-fat-diet model was used. Administration of high-fat diet exacerbated UVB-induced inflammation. Administration of the high-fat diet enhanced β-catenin signaling and the levels of its downstream targets (c-Myc, cyclin D1, cyclin D2, MMP-2 and MMP-9) in UVB-exposed skin and skin tumors in SKH-1 mice. These data suggest that UV-induced COX-2/PGE2 stimulates β-catenin signaling, and that β-catenin activation may contribute to skin carcinogenesis. PMID:24481495

  2. 4-Aminoethylamino-emodin – a novel potent inhibitor of GSK-3β– acts as an insulin-sensitizer avoiding downstream effects of activated β-catenin

    PubMed Central

    Gebhardt, Rolf; Lerche, Katja S; Götschel, Frank; Günther, Robert; Kolander, Jens; Teich, Lars; Zellmer, Sebastian; Hofmann, Hans-Jörg; Eger, Kurt; Hecht, Andreas; Gaunitz, Frank

    2010-01-01

    Abstract Glycogen synthase kinase-3β (GSK-3β) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3β with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3β inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3β inhibitors (SB216763 and LiCl) or Wnt-3α-conditioned medium, however, L4 does not induce reporter and target genes of activated β-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates β-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by β-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3β inhibition but avoiding hazardous effects such as activation of β-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer. PMID:19228266

  3. Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling.

    PubMed

    Balagopalan, Lakshmi; Barr, Valarie A; Kortum, Robert L; Park, Anna K; Samelson, Lawrence E

    2013-04-15

    A controversy has recently emerged regarding the location of the cellular pool of the adapter linker for activation of T cells (LAT) that participates in propagation of signals downstream of the TCR. In one model phosphorylation and direct recruitment of cell surface LAT to activation-induced microclusters is critical for T cell activation, whereas in the other model vesicular, but not surface, LAT participates in these processes. By using a chimeric version of LAT that can be tracked via an extracellular domain, we provide evidence that LAT located at the cell surface can be recruited efficiently to activation-induced microclusters within seconds of TCR engagement. Importantly, we also demonstrate that this pool of LAT at the plasma membrane is rapidly phosphorylated. Our results provide support for the model in which the cell utilizes LAT from the cell surface for rapid responses to TCR stimulation. PMID:23487428

  4. A Phosphatase Activity of Sts-1 Contributes to the Suppression of TCR Signaling

    SciTech Connect

    Mikhailik,A.; Ford, B.; Keller, J.; Chen, Y.; Nassar, N.; Carpino, N.

    2007-01-01

    Precise signaling by the T cell receptor (TCR) is crucial for a proper immune response. To ensure that T cells respond appropriately to antigenic stimuli, TCR signaling pathways are subject to multiple levels of regulation. Sts-1 negatively regulates signaling pathways downstream of the TCR by an unknown mechanism(s). Here, we demonstrate that Sts-1 is a phosphatase that can target the tyrosine kinase Zap-70 among other proteins. The X-ray structure of the Sts-1 C terminus reveals that it has homology to members of the phosphoglycerate mutase/acid phosphatase (PGM/AcP) family of enzymes, with residues known to be important for PGM/AcP catalytic activity conserved in nature and position in Sts-1. Point mutations that impair Sts-1 phosphatase activity in vitro also impair the ability of Sts-1 to regulate TCR signaling in T cells. These observations reveal a PGM/AcP-like enzyme activity involved in the control of antigen receptor signaling.

  5. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  6. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity

    PubMed Central

    Tarayrah, Lama; Li, Yuping; Gan, Qiang; Chen, Xin

    2015-01-01

    ABSTRACT Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. Here we report that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid) maintains germline stem cell (GSC) mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Our findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. Our study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities. PMID:26490676

  7. Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

    NASA Technical Reports Server (NTRS)

    Kumel, Yasuhiro; Shimokawa, Hitoyata; Morita, Sadao; Katano, Hisako; Akiyama, Hideo; Hirano, Masahiko; Ohya, Keiichi; Sams, Clarence F.; Whitson, Peggy A.

    2005-01-01

    Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

  8. Human ECG signal parameters estimation during controlled physical activity

    NASA Astrophysics Data System (ADS)

    Maciejewski, Marcin; Surtel, Wojciech; Dzida, Grzegorz

    2015-09-01

    ECG signal parameters are commonly used indicators of human health condition. In most cases the patient should remain stationary during the examination to decrease the influence of muscle artifacts. During physical activity, the noise level increases significantly. The ECG signals were acquired during controlled physical activity on a stationary bicycle and during rest. Afterwards, the signals were processed using a method based on Pan-Tompkins algorithms to estimate their parameters and to test the method.

  9. Mathematical modeling of light-mediated HPA axis activity and downstream implications on the entrainment of peripheral clock genes.

    PubMed

    Mavroudis, Panteleimon D; Corbett, Siobhan A; Calvano, Steven E; Androulakis, Ioannis P

    2014-10-15

    In this work we propose a semimechanistic model that describes the photic signal transduction to the hypothalamic-pituitary-adrenal (HPA) axis that ultimately regulates the synchronization of peripheral clock genes (PCGs). Our HPA axis model predicts that photic stimulation induces a type-1 phase response curve to cortisol's profile with increased cortisol sensitivity to light exposure in its rising phase, as well as the shortening of cortisol's period as constant light increases (Aschoff's first rule). Furthermore, our model provides insight into cortisol's phase and amplitude dependence on photoperiods and reveals that cortisol maintains highest amplitude variability when it is entrained by a balanced schedule of light and dark periods. Importantly, by incorporating the links between HPA axis and PCGs we were able to investigate how cortisol secretion impacts the entrainment of a population of peripheral cells and show that disrupted light schedules, leading to blunted cortisol secretion, fail to synchronize a population of PCGs which further signifies the loss of circadian rhythmicity in the periphery of the body. PMID:25073602

  10. Dact2 Represses PITX2 Transcriptional Activation and Cell Proliferation through Wnt/beta-Catenin Signaling during Odontogenesis

    PubMed Central

    Li, Xiao; Florez, Sergio; Wang, Jianbo; Cao, Huojun; Amendt, Brad A.

    2013-01-01

    Dact proteins belong to the Dapper/Frodo protein family and function as cytoplasmic attenuators in Wnt and TGFβ signaling. Previous studies show that Dact1 is a potent Wnt signaling inhibitor by promoting degradation of β-catenin. We report a new mechanism for Dact2 function as an inhibitor of the canonical Wnt signaling pathway by interacting with PITX2. PITX2 is a downstream transcription factor in Wnt/β-catenin signaling, and PITX2 synergizes with Lef-1 to activate downstream genes. Immunohistochemistry verified the expression of Dact2 in the tooth epithelium, which correlated with Pitx2 epithelial expression. Dact2 loss of function and PITX2 gain of function studies reveal a feedback mechanism for controlling Dact2 expression. Pitx2 endogenously activates Dact2 expression and Dact2 feeds back to repress Pitx2 transcriptional activity. A Topflash reporter system was employed showing PITX2 activation of Wnt signaling, which is attenuated by Dact2. Transient transfections demonstrate the inhibitory effect of Dact2 on critical dental epithelial differentiation factors during tooth development. Dact2 significantly inhibits PITX2 activation of the Dlx2 and amelogenin promoters. Multiple lines of evidence conclude the inhibition is achieved by the physical interaction between Dact2 and Pitx2 proteins. The loss of function of Dact2 also reveals increased cell proliferation due to up-regulated Wnt downstream genes, cyclinD1 and cyclinD2. In summary, we have identified a novel role for Dact2 as an inhibitor of the canonical Wnt pathway in embryonic tooth development through its regulation of cell proliferation and differentiation. PMID:23349981

  11. Oxidative Stress–Induced JNK1/2 Activation Triggers Proapoptotic Signaling and Apoptosis That Leads to Diabetic Embryopathy

    PubMed Central

    Li, Xuezheng; Weng, Hongbo; Xu, Cheng; Reece, E. Albert; Yang, Peixin

    2012-01-01

    Oxidative stress and apoptosis are implicated in the pathogenesis of diabetic embryopathy. The proapoptotic c-Jun NH2-terminal kinases (JNK)1/2 activation is associated with diabetic embryopathy. We sought to determine whether 1) hyperglycemia-induced oxidative stress is responsible for the activation of JNK1/2 signaling, 2) JNK1 contributes to the teratogenicity of hyperglycemia, and 3) both JNK1 and JNK2 activation cause activation of downstream transcription factors, caspase activation, and apoptosis, resulting in neural tube defects (NTDs). Wild-type (WT) embryos from nondiabetic WT dams and WT, superoxide dismutase (SOD)1–overexpressing, jnk1+/−, jnk1−/−, and jnk2−/− embryos exposed to maternal hyperglycemia were used to assess JNK1/2 activation, NTDs, activation of transcription factors downstream of JNK1/2, caspase cascade, and apoptosis. SOD1 overexpression abolished diabetes-induced activation of JNK1/2 and their downstream effectors: phosphorylation of c-Jun, activating transcription factor 2, and E twenty-six–like transcription factor 1 and dephosphorylation of forkhead box class O3a. jnk1−/− embryos had significantly lower incidences of NTDs than those of WT or jnk1+/− embryos. Either jnk1 or jnk2 gene deletion blocked diabetes-induced activation of JNK1/2 signaling, caspases 3 and 8, and apoptosis in Sox1+ neural progenitors of the developing neural tube. Our results show that JNK1 and JNK2 are equally involved in diabetic embryopathy and that the oxidative stress–JNK1/2–caspase pathway mediates the proapoptotic signals and the teratogenicity of maternal diabetes. PMID:22688338

  12. Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

    PubMed Central

    Roybal, Kole T.; Mace, Emily M.; Clark, Danielle J.; Leard, Alan D.; Herman, Andrew; Verkade, Paul; Orange, Jordan S.; Wülfing, Christoph

    2015-01-01

    Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation. PMID:26237588

  13. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus.

    PubMed

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  14. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus

    PubMed Central

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  15. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction.

    PubMed

    Beguerisse-Díaz, Mariano; Desikan, Radhika; Barahona, Mauricio

    2016-08-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction. PMID:27581482

  16. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction

    PubMed Central

    Desikan, Radhika

    2016-01-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction. PMID:27581482

  17. Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense*

    PubMed Central

    Jones, Alexis; Kainz, Danielle; Khan, Faatima; Lee, Cara; Carrithers, Michael D.

    2014-01-01

    Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon β. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation. PMID:25368329

  18. Calmodulin activity regulates group I metabotropic glutamate receptor-mediated signal transduction and synaptic depression.

    PubMed

    Sethna, Ferzin; Zhang, Ming; Kaphzan, Hanoch; Klann, Eric; Autio, Dawn; Cox, Charles L; Wang, Hongbing

    2016-05-01

    Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR 5 (mGluR1/5), are coupled to Gq and modulate activity-dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation-dependent long-term depression (LTD). Although it has been established that intracellular Ca(2+) and the Gq-regulated signaling molecules are required for mGluR1/5 LTD, whether and how Ca(2+) regulates Gq signaling and upregulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of the Ca(2+) sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor N-[4-aminobutyl]-5-chloro-2-naphthalenesulfonamide hydrochloride (W13) suppressed the mGluR1/5-stimulated activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p70-S6 kinase 1 (S6K1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist-induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca(2+) /CaM-dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Furthermore, disruption of the CaM-CaMK-ERK1/2 signaling cascade suppressed the mGluR1/5-stimulated upregulation of Arc expression. Altogether, our data suggest CaM as a new Gq signaling component for coupling Ca(2+) and protein upregulation and regulating mGluR1/5-mediated synaptic modification. PMID:26864654

  19. Canonical transient receptor potential channels promote cardiomyocyte hypertrophy through activation of calcineurin signaling.

    PubMed

    Bush, Erik W; Hood, David B; Papst, Philip J; Chapo, Joseph A; Minobe, Wayne; Bristow, Michael R; Olson, Eric N; McKinsey, Timothy A

    2006-11-01

    The calcium/calmodulin-dependent phosphatase calcineurin plays a central role in the control of cardiomyocyte hypertrophy in response to pathological stimuli. Although calcineurin is present at high levels in normal heart, its activity appears to be unaffected by calcium during the course of a cardiac cycle. The mechanism(s) whereby calcineurin is selectively activated by calcium under pathological conditions has remained unclear. Here, we demonstrate that diverse signals for cardiac hypertrophy stimulate expression of canonical transient receptor potential (TRPC) channels. TRPC consists of a family of seven membrane-spanning nonselective cation channels that have been implicated in the nonvoltage-gated influx of calcium in response to G protein-coupled receptor signaling, receptor tyrosine kinase signaling, and depletion of internal calcium stores. TRPC3 expression is up-regulated in multiple rodent models of pathological cardiac hypertrophy, whereas TRPC5 expression is induced in failing human heart. We demonstrate that TRPC promotes cardiomyocyte hypertrophy through activation of calcineurin and its downstream effector, the nuclear factor of activated T cells transcription factor. These results define a novel role for TRPC channels in the control of cardiac growth, and suggest that a TRPC-derived pool of calcium contributes to selective activation of calcineurin in diseased heart. PMID:16950785

  20. A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

    PubMed Central

    Liu, Yunhao; Collins, Caitlin; Kiosses, William B.; Murray, Ann M.; Joshi, Monika; Shepherd, Tyson R.; Fuentes, Ernesto J.

    2013-01-01

    Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase. PMID:23733346

  1. Chemical downstream etching of tungsten

    SciTech Connect

    Blain, M.G.; Jarecki, R.L.; Simonson, R.J.

    1998-07-01

    The downstream etching of tungsten and tungsten oxide has been investigated. Etching of chemical vapor deposited tungsten and e-beam deposited tungsten oxide samples was performed using atomic fluorine generated by a microwave discharge of argon and NF{sub 3}. Etching was found to be highly activated with activation energies approximated to be 6.0{plus_minus}0.5thinspkcal/mol and 5.4{plus_minus}0.4thinspkcal/mol for W and WO{sub 3}, respectively. In the case of F etching of tungsten, the addition of undischarged nitric oxide (NO) directly into the reaction chamber results in the competing effects of catalytic etch rate enhancement and the formation of a nearly stoichiometric WO{sub 3} passivating tungsten oxide film, which ultimately stops the etching process. For F etching of tungsten oxide, the introduction of downstream NO reduces the etch rate. {copyright} {ital 1998 American Vacuum Society.}

  2. Exposure histories derived from selenium in otoliths of three cold-water fish species captured downstream from coal mining activity.

    PubMed

    Friedrich, Lisa A; Orr, Patricia L; Halden, Norman M; Yang, Panseok; Palace, Vince P

    2011-10-01

    Establishing exposure to contaminants within a given environment is often difficult for fish species with large home ranges. Chemical analyses of muscle or visceral tissue are useful indicators of recent exposure, but depuration, metabolic transformation, and tissue redistributions can alter temporal resolution. Otoliths are metabolically stable and thus provide complete chemical records within their calcified tissues that, when coupled to the annular structure, can provide temporal resolution for exposure to trace metals. Otoliths from bull trout, cutthroat trout, and mountain whitefish from an area rich in seleniferous soils and with active coal mining activity were analyzed for selenium to determine any history of exposure to elevated levels of selenium. Selenium concentrations in otolith primordia tended to be low, indicating that these fish emerged in low selenium areas. Later life stages showed peaks of high Se concentrations, suggesting that individuals moved into areas of increased selenium later in life. Individuals captured from the same area had a wide variety of selenium exposure profiles, indicating that these fish do not move en masse into and out of high-selenium areas. Year-to-year variability of selenium exposure patterns within an otolith suggests inconsistent utilization of high- and low-selenium areas by the individual. The inconsistent exposure profiles for these fish, in addition to their home range of tens of kilometres, indicate that soft tissue concentrations, while useful indicators of recent exposure, cannot be relied upon to provide a life history recording of exposure. PMID:21899824

  3. Atypical nuclear apoptosis downstream to caspase-3 activation in ara-C treated CCRF-CEM cells.

    PubMed

    Badran, Adel; Iwasaki, Hiromichi; Inoue, Hitoshi; Ueda, Takanori

    2003-03-01

    The necessity of internucleosomal DNA fragmentation for the complete execution of apoptosis is still controversial. While investigating the apoptotic pathway induced by 1-beta-D-arabinofuranosylcytosine (ara-C) in the human T-lymphoblastic leukemia CCRF-CEM (CEM) cells, we could easily retrieve high molecular weight (HMW) DNA fragments with a predominant size of 50 kb. However, under the same circumstances, internucleosomal DNA fragmentation characteristic of apoptosis was undetectable despite estimated heightened caspase-3 activity. Paradoxically, generation of low molecular weight DNA fragments was readily demonstrable by flow cytometric and immunohistochemical evidence in the absence of any detectable DNA ladder formation. These findings present a proof that, within certain contexts, small-sized DNA fragmentation occurring in apoptosis may not necessarily be of the ladder yielding internucleosomal integer multiples' pattern. Our data also add to the evidence that the machinery underlying HMW DNA fragmentation is distinct from that responsible for the internucleosomal one. PMID:12579303

  4. The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

    PubMed Central

    Kopan, Raphael; Ilagan, Ma. Xenia G.

    2009-01-01

    Notch signaling regulates many aspects of metazoan development and tissue renewal. Accordingly, misregulation or loss of Notch signaling underlies multiple human disorders, from developmental syndromes to adult onset diseases and cancer. Notch receptor activation is irreversible as it involves proteolysis-mediated release of the Notch intracellular domain, translocation to the nucleus, and association with a DNA-bound protein. Even though each Notch molecule signals only once without amplification by secondary messenger cascades, Notch signaling is remarkably robust in most tissues. In this review, we highlight the recent studies that reveal new molecular details involved in regulating ligand-mediated activation, receptor proteolysis and target selection. PMID:19379690

  5. Rhamnazin, a novel inhibitor of VEGFR2 signaling with potent antiangiogenic activity and antitumor efficacy

    SciTech Connect

    Yu, Yao; Cai, Wei; Pei, Chong-gang; Shao, Yi

    2015-03-20

    Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer therapy. The identification of new drugs from natural products has a long and successful history. In this study, we described a novel VEGFR2 inhibitor, rhamnazin, which inhibits tumor angiogenesis and growth. Rhamnazin significantly inhibited proliferation, migration and tube formation of human umbilical vascular endothelial cells (HUVECs) in vitro as well as inhibited sprouts formation of rat aorta ring. In addition, it inhibited vascular endothelial growth factor (VEGF)-induced phosphorylation of VEGFR2 and its downstream signaling regulator in HUVECs. Moreover, rhamnazin could directly inhibit proliferation of breast cancer cells MDA-MB-231 in vitro and in vivo. Oral administration of rhamnazin at a dose of 200 mg/kg/day could markedly inhibited human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that rhamnazin inhibits angiogenesis and may be a promising anticancer drug candidate. - Highlights: • Rhamnazin inhibits the response of HUVECs to VEGF in vitro. • Rhamnazin inhibits VEGFR2 kinase activity and its downstream signaling. • Rhamnazin prevents the growth of MDA-MB-231 tumor and reduces micro-vessel density in vivo.

  6. Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor {gamma}, controls hepatitis B virus replication

    SciTech Connect

    Yoon, Sarah; Jung, Jaesung; Kim, Taeyeung; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

    2011-01-20

    In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein {alpha} (C/EBP{alpha}), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), adiponectin, liver X receptor {alpha} (LXR{alpha}), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPAR{gamma} and C/EBP{alpha}. Conversely, HBV replication was upregulated by adiponectin and PPAR{gamma} agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.

  7. Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation.

    PubMed

    Li, Yong D; Block, Edward R; Patel, Jawaharlal M

    2002-10-01

    Signaling events involving angiotensin IV (ANG IV)-mediated pulmonary artery endothelial cell (PAEC) proliferation were examined. ANG IV significantly increased upstream phosphatidylinositide (PI) 3-kinase (PI3K), PI-dependent kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), and protein kinase B-alpha/Akt (PKB-alpha) activities, as well as downstream p70 ribosomal S6 kinase (p70S6K) activities and/or phosphorylation of these proteins. ANG IV also significantly increased 5-bromo-2'-deoxy-uridine incorporation into newly synthesized DNA in a concentration- and time-dependent manner. Pretreatment of cells with wortmannin and LY-294002, inhibitors of PI3K, or rapamycin, an inhibitor of the mammalian target of rapamycin kinase and p70S6K, diminished the ANG IV-mediated activation of PDK-1 and PKB-alpha as well as phosphorylation of p70S6K. Although an inhibitor of mitogen-activated protein kinase kinase, PD-98059, but not rapamycin, blocked ANG IV-induced phosphorylation of ERK1/2, both PD-98059 and rapamycin independently caused partial reduction in ANG IV-mediated cell proliferation. However, simultaneous treatment with PD-98059 and rapamycin resulted in total inhibition of ANG IV-induced cell proliferation. These results demonstrate that ANG IV-induced DNA synthesis is regulated in a coordinated fashion involving multiple signaling modules in PAEC. PMID:12225947

  8. Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine mammary epithelial cells contribute to the innate immune response to intramammary infections by recognizing pathogens through specialized pattern recognition receptors. Toll-like receptor 4 (TLR4) is one such receptor that binds and is activated by lipopolysaccharide (LPS), a component of the...

  9. Synapsin III Acts Downstream of Semaphorin 3A/CDK5 Signaling to Regulate Radial Migration and Orientation of Pyramidal Neurons In Vivo

    PubMed Central

    Perlini, Laura E.; Szczurkowska, Joanna; Ballif, Bryan A.; Piccini, Alessandra; Sacchetti, Silvio; Giovedì, Silvia; Benfenati, Fabio; Cancedda, Laura

    2015-01-01

    Summary Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5) phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation. PMID:25843720

  10. Kresoxim-methyl primes Medicago truncatula plants against abiotic stress factors via altered reactive oxygen and nitrogen species signalling leading to downstream transcriptional and metabolic readjustment.

    PubMed

    Filippou, Panagiota; Antoniou, Chrystalla; Obata, Toshihiro; Van Der Kelen, Katrien; Harokopos, Vaggelis; Kanetis, Loukas; Aidinis, Vassilis; Van Breusegem, Frank; Fernie, Alisdair R; Fotopoulos, Vasileios

    2016-03-01

    Biotic and abiotic stresses, such as fungal infection and drought, cause major yield losses in modern agriculture. Kresoxim-methyl (KM) belongs to the strobilurins, one of the most important classes of agricultural fungicides displaying a direct effect on several plant physiological and developmental processes. However, the impact of KM treatment on salt and drought stress tolerance is unknown. In this study we demonstrate that KM pre-treatment of Medicago truncatula plants results in increased protection to drought and salt stress. Foliar application with KM prior to stress imposition resulted in improvement of physiological parameters compared with stressed-only plants. This protective effect was further supported by increased proline biosynthesis, modified reactive oxygen and nitrogen species signalling, and attenuation of cellular damage. In addition, comprehensive transcriptome analysis identified a number of transcripts that are differentially accumulating in drought- and salinity-stressed plants (646 and 57, respectively) after KM pre-treatment compared with stressed plants with no KM pre-treatment. Metabolomic analysis suggests that the priming role of KM in drought- and to a lesser extent in salinity-stressed plants can be attributed to the regulation of key metabolites (including sugars and amino acids) resulting in protection against abiotic stress factors. Overall, the present study highlights the potential use of this commonly used fungicide as a priming agent against key abiotic stress conditions. PMID:26712823

  11. Kresoxim-methyl primes Medicago truncatula plants against abiotic stress factors via altered reactive oxygen and nitrogen species signalling leading to downstream transcriptional and metabolic readjustment

    PubMed Central

    Filippou, Panagiota; Antoniou, Chrystalla; Obata, Toshihiro; Van Der Kelen, Katrien; Harokopos, Vaggelis; Kanetis, Loukas; Aidinis, Vassilis; Van Breusegem, Frank; Fernie, Alisdair R; Fotopoulos, Vasileios

    2016-01-01

    Biotic and abiotic stresses, such as fungal infection and drought, cause major yield losses in modern agriculture. Kresoxim-methyl (KM) belongs to the strobilurins, one of the most important classes of agricultural fungicides displaying a direct effect on several plant physiological and developmental processes. However, the impact of KM treatment on salt and drought stress tolerance is unknown. In this study we demonstrate that KM pre-treatment of Medicago truncatula plants results in increased protection to drought and salt stress. Foliar application with KM prior to stress imposition resulted in improvement of physiological parameters compared with stressed-only plants. This protective effect was further supported by increased proline biosynthesis, modified reactive oxygen and nitrogen species signalling, and attenuation of cellular damage. In addition, comprehensive transcriptome analysis identified a number of transcripts that are differentially accumulating in drought- and salinity-stressed plants (646 and 57, respectively) after KM pre-treatment compared with stressed plants with no KM pre-treatment. Metabolomic analysis suggests that the priming role of KM in drought- and to a lesser extent in salinity-stressed plants can be attributed to the regulation of key metabolites (including sugars and amino acids) resulting in protection against abiotic stress factors. Overall, the present study highlights the potential use of this commonly used fungicide as a priming agent against key abiotic stress conditions. PMID:26712823

  12. Diacylglycerol kinase ζ limits B cell antigen receptor-dependent activation of ERK signaling to inhibit early antibody responses.

    PubMed

    Wheeler, Matthew L; Dong, Matthew B; Brink, Robert; Zhong, Xiao-Ping; DeFranco, Anthony L

    2013-10-15

    Signaling downstream of the B cell antigen receptor (BCR) is tightly regulated to enable cells to gauge the strength and duration of antigen-receptor interactions and to respond appropriately. We investigated whether metabolism of the second messenger diacylglycerol (DAG) by members of the family of DAG kinases (DGKs) played a role in modulating the magnitude of signaling by DAG downstream of the BCR. In the absence of DGKζ, the threshold for BCR signaling, measured as activation of the Ras-extracellular signal-regulated kinase (ERK) pathway, was markedly reduced in mature follicular B cells, which resulted in enhanced responses to antigen in vitro and in vivo. Inhibition of DAG signaling by DGKζ limited the number of antibody-secreting cells that were generated early in response to T cell-independent type 2 antigens, as well as to T cell-dependent antigens. Furthermore, the effect of loss of DGKζ closely resembled the effect of increasing the affinity of the BCR for antigen during the T cell-dependent antibody response. These results suggest that the magnitude of DAG signaling is important for translating the affinity of the BCR for antigen into the amount of antibody produced during the early stages of an immune response. PMID:24129701

  13. Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling.

    PubMed

    Raufman, Jean-Pierre; Shant, Jasleen; Guo, Chang Yue; Roy, Sanjit; Cheng, Kunrong

    2008-05-01

    Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation. PMID:18064605

  14. Activation of diverse signaling pathways by oncogenic PIK3CA mutations

    PubMed Central

    Wu, Xinyan; Renuse, Santosh; Sahasrabuddhe, Nandini A.; Zahari, Muhammad Saddiq; Chaerkady, Raghothama; Kim, Min-Sik; Nirujogi, Raja S.; Mohseni, Morassa; Kumar, Praveen; Raju, Rajesh; Zhong, Jun; Yang, Jian; Neiswinger, Johnathan; Jeong, Jun-Seop; Newman, Robert; Powers, Maureen A.; Somani, Babu Lal; Gabrielson, Edward; Sukumar, Saraswati; Stearns, Vered; Qian, Jiang; Zhu, Heng; Vogelstein, Bert; Park, Ben Ho; Pandey, Akhilesh

    2014-01-01

    The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signaling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signaling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signaling events and for discovering novel signaling molecules as readouts of pathway activation or potential therapeutic targets. PMID:25247763

  15. Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity.

    PubMed

    Bülow, Margret H; Bülow, Torsten R; Hoch, Michael; Pankratz, Michael J; Jünger, Martin A

    2014-01-01

    Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells. PMID:24513978

  16. Neutrino Factory Downstream Systems

    SciTech Connect

    Zisman, Michael S.

    2009-12-23

    We describe the Neutrino Factory accelerator systems downstream from the target and capture area. These include the bunching and phase rotation, cooling, acceleration, and decay ring systems. We also briefly discuss the R&D program under way to develop these systems, and indicate areas where help from CERN would be invaluable.

  17. Integrin endosomal signalling suppresses anoikis.

    PubMed

    Alanko, Jonna; Mai, Anja; Jacquemet, Guillaume; Schauer, Kristine; Kaukonen, Riina; Saari, Markku; Goud, Bruno; Ivaska, Johanna

    2015-11-01

    Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis. PMID:26436690

  18. Apelin-APJ Signaling is a Critical Regulator of Endothelial MEF2 Activation in Cardiovascular Development

    PubMed Central

    Kang, Yujung; Kim, Jongmin; Anderson, Joshua P.; Wu, Jingxia; Gleim, Scott R.; Kundu, Ramendra K.; McLean, Danielle L.; Kim, Jun-dae; Park, Hyekyung; Jin, Suk-won; Hwa, John; Quertermous, Thomas; Chun, Hyung J.

    2013-01-01

    Rationale The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. Objective This study aimed to characterize the embryonic lethal phenotype of the Apj−/− mice and define the involved downstream signaling targets. Methods and Results We report the first characterization of the embryonic lethality of the Apj−/− mice. Greater than half of the expected Apj−/− embryos died in utero due to cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj−/− embryos surviving to later stages demonstrated incomplete vascular maturation due to a deficiency of vascular smooth muscle cells, and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, non-canonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of MEF2 (myocyte enhancer factor 2). Apj−/− mice have greater endocardial Hdac4 and Hdac5 nuclear localization, and reduced expression of the MEF2 transcriptional target Klf2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj−/− embryos and endothelial cells. Conclusions Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system. PMID:23603510

  19. IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling.

    PubMed

    Lazear, Helen M; Lancaster, Alissa; Wilkins, Courtney; Suthar, Mehul S; Huang, Albert; Vick, Sarah C; Clepper, Lisa; Thackray, Larissa; Brassil, Margaret M; Virgin, Herbert W; Nikolich-Zugich, Janko; Moses, Ashlee V; Gale, Michael; Früh, Klaus; Diamond, Michael S

    2013-01-01

    Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5. PMID:23300459

  20. Wnt Signaling in Neurogenesis during Aging and Physical Activity

    PubMed Central

    Chen, Michael; Do, Huong

    2012-01-01

    Over the past decade, much progress has been made regarding our understanding of neurogenesis in both young and old animals and where it occurs throughout the lifespan, although the growth of new neurons declines with increasing age. In addition, physical activity can reverse this age-dependent decline in neurogenesis. Highly correlated with this decline is the degree of inter and intracellular Wnt signaling, the molecular mechanisms of which have only recently started to be elucidated. So far, most of what we know about intracellular signaling during/following exercise centers around the CREB/CRE initiated transcriptional events. Relatively little is known, however, about how aging and physical activity affect the Wnt signaling pathway. Herein, we briefly review the salient features of neurogenesis in young and then in old adult animals. Then, we discuss Wnt signaling and review the very few in vitro and in vivo studies that have examined the Wnt signaling pathways in aging and physical activity. PMID:24961268

  1. Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability

    PubMed Central

    Tsai, Jia-Shiuan; Chao, Cheng-Han; Lin, Lih-Yuan

    2016-01-01

    Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. PMID:26751215

  2. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling.

    PubMed

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-25

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol. PMID:27545348

  3. IRF-3, IRF-5, and IRF-7 Coordinately Regulate the Type I IFN Response in Myeloid Dendritic Cells Downstream of MAVS Signaling

    PubMed Central

    Lazear, Helen M.; Lancaster, Alissa; Wilkins, Courtney; Suthar, Mehul S.; Huang, Albert; Vick, Sarah C.; Clepper, Lisa; Thackray, Larissa; Brassil, Margaret M.; Virgin, Herbert W.; Nikolich-Zugich, Janko; Moses, Ashlee V.; Gale, Michael; Früh, Klaus; Diamond, Michael S.

    2013-01-01

    Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3−/−×Irf7−/− double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3−/−×Irf5−/−×Irf7−/− triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar−/−). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar−/− mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs−/− mDC. The relative equivalence of TKO and Mavs−/− responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5. PMID:23300459

  4. Activation of endothelial β-catenin signaling induces heart failure

    PubMed Central

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  5. Activation of endothelial β-catenin signaling induces heart failure.

    PubMed

    Nakagawa, Akito; Naito, Atsuhiko T; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P; Adams, Ralf H; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  6. A conserved Six-Eya cassette acts downstream of Wnt signaling to direct non-myogenic versus myogenic fates in the C. elegans postembryonic mesoderm.

    PubMed

    Amin, Nirav M; Lim, Sung-Eun; Shi, Herong; Chan, Tiffany L; Liu, Jun

    2009-07-15

    The subdivision of mesodermal cells into muscle and non-muscle cells is crucial to animal development. In the C. elegans postembryonic mesoderm, this subdivision is a result of an asymmetric cell division that leads to the formation of striated body wall muscles and non-muscle coelomocytes. Here we report that the Six homeodomain protein CEH-34 and its cofactor Eyes Absent, EYA-1, function synergistically to promote the non-muscle fate in cells also competent to form muscles. We further show that the asymmetric expression of ceh-34 and eya-1 is regulated by a combination of 1) mesodermal intrinsic factors MAB-5, HLH-1 and FOZI-1, 2) differential POP-1 (TCF/LEF) transcriptional activity along the anterior-posterior axis, and 3) coelomocyte competence factor(s). These factors are conserved in both vertebrates and invertebrates, suggesting a conserved paradigm for mesoderm development in metazoans. PMID:19427847

  7. Eccentric Exercise Activates Novel Transcriptional Regulation of Hypertrophic Signaling Pathways Not Affected by Hormone Changes

    PubMed Central

    MacNeil, Lauren G.; Melov, Simon; Hubbard, Alan E.; Baker, Steven K.; Tarnopolsky, Mark A.

    2010-01-01

    Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17β-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2. PMID:20502695

  8. Extracellular signal-regulated kinase (ERK) activation is required for porcine epidemic diarrhea virus replication.

    PubMed

    Kim, Youngnam; Lee, Changhee

    2015-10-01

    Porcine epidemic diarrhea virus (PEDV) is a highly enteropathogenic coronavirus of swine that causes acute enteritis with high mortality in nursery piglets. To date, the cellular factors involved in PEDV replication have not been well defined. The extracellular signal-regulated kinase (ERK) that serves as a critical component of cellular signal transduction pathways to modulate a variety of cellular functions has been shown to regulate several viral infections. In the present study, we found that PEDV activates ERK1/2 early in infection independently of viral replication. The PEDV-induced ERK1/2 activation resulted in the phosphorylation of its downstream substrate Elk-1 in infected cells. Treatment with ERK inhibitors or ERK1/2 knockdown significantly suppressed viral progeny production. Inhibition of ERK activation also diminished viral protein expression and genomic and subgenomic RNA transcription. These findings indicate that the ERK signaling pathway plays an important role in the PEDV life cycle and beneficially contributes to viral infection. PMID:26115165

  9. Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

    SciTech Connect

    Kim, Se-Hee; Schmitt, Christopher E.; Woolls, Melissa J.; Holland, Melinda B.; Kim, Jun-Dae; Jin, Suk-Won

    2013-01-25

    Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.

  10. Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.

    PubMed

    Lee, Jinyong; Ghil, Sungho

    2016-05-01

    Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition. PMID:26829215

  11. A novel c-Jun-dependent signal transduction pathway necessary for the transcriptional activation of interferon gamma response genes.

    PubMed

    Gough, Daniel J; Sabapathy, Kanaga; Ko, Enoch Yi-No; Arthur, Helen A; Schreiber, Robert D; Trapani, Joseph A; Clarke, Christopher J P; Johnstone, Ricky W

    2007-01-12

    The biological effects of interferon gamma (IFNgamma) are mediated by interferon-stimulated genes (ISGs), many of which are activated downstream of Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling. Herein we have shown that IFNgamma rapidly activated AP-1 DNA binding that required c-Jun but was independent of JAK1 and STAT1. IFNgamma-induced c-Jun phosphorylation and AP-1 DNA binding required the MEK1/2 and ERK1/2 signaling pathways, whereas the JNK1/2 and p38 mitogen-activated protein kinase pathways were dispensable. The induction of several ISGs, including ifi-205 and iNOS, was impaired in IFNgamma-treated c-Jun-/- cells, but others, such as IP-10 and SOCS3, were unaffected, and chromatin immunoprecipitation demonstrated that c-Jun binds to the iNOS promoter following treatment with IFNgamma. Thus, IFNgamma induced JAK1- and STAT1-independent activation of the ERK mitogen-activated protein kinase pathway, phosphorylation of c-Jun, and activation of AP-1 DNA binding, which are important for the induction of a subset of ISGs. This represents a novel signal transduction pathway induced by IFNgamma that proceeds in parallel with conventional JAK/STAT signaling to activate ISGs. PMID:17105733

  12. RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

    PubMed Central

    Li, Luowei; Kedei, Noemi; Tóth, Zsuzsanna E.; Czap, Alexandra; Velasquez, Julia F.; Mihova, Daniela; Michalowski, Aleksandra M.; Yuspa, Stuart H.; Blumberg, Peter M.

    2014-01-01

    RasGRP3, an activator for H-Ras, R-Ras and Rap1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from HGF or EGF stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation, and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target. PMID:21602881

  13. Autocrine activity of BDNF induced by the STAT3 signaling pathway causes prolonged TrkB activation and promotes human non-small-cell lung cancer proliferation

    PubMed Central

    Chen, Bo; Liang, Yan; He, Zheng; An, Yunhe; Zhao, Weihong; Wu, Jianqing

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily, which has been implicated in the pathophysiology of the nervous system. Recently, several studies have suggested that BDNF and/or its receptor, tropomyosin related kinase B (TrkB), are involved in tumor growth and metastasis in several cancers, including prostate cancer, neuroblastoma, pancreatic ductal carcinoma, hepatocellular carcinoma, and lung cancer. Despite the increasing emphasis on BDNF/TrkB signaling in human tumors, how it participates in primary tumors has not yet been determined. Additionally, little is known about the molecular mechanisms that elicit signaling downstream of TrkB in the progression of non-small-cell lung cancer (NSCLC). In this study, we report the significant expression of BDNF in NSCLC samples and show that BDNF stimulation increases the synthesis of BDNF itself through activation of STAT3 in lung cancer cells. The release of BDNF can in turn activate TrkB signaling. The activation of both TrkB and STAT3 contribute to downstream signaling and promote human non-small-cell lung cancer proliferation. PMID:27456333

  14. Quantifying bursting neuron activity from calcium signals using blind deconvolution.

    PubMed

    Park, In Jun; Bobkov, Yuriy V; Ache, Barry W; Principe, Jose C

    2013-09-15

    Advances in calcium imaging have enabled studies of the dynamic activity of both individual neurons and neuronal assemblies. However, challenges, such as unknown nonlinearities in the spike-calcium relationship, noise, and the often relatively low temporal resolution of the calcium signal compared to the time-scale of spike generation, restrict the accurate estimation of action potentials from the calcium signal. Complex neuronal discharge, such as the activity demonstrated by bursting and rhythmically active neurons, represents an even greater challenge for reconstructing spike trains based on calcium signals. We propose a method using blind calcium signal deconvolution based on an information-theoretic approach. This model is meant to maximise the output entropy of a nonlinear filter where the nonlinearity is defined by the cumulative distribution function of the spike signal. We tested our maximum entropy (ME) algorithm using bursting olfactory receptor neurons (bORNs) of the lobster olfactory organ. The advantage of the ME algorithm is that the filter can be trained online based only on the statistics of the spike signal, without any assumptions regarding the unknown transfer function characterizing the relation between the spike and calcium signal. We show that the ME method is able to more accurately reconstruct the timing of the first and last spikes of a burst compared to other methods and that it improves the temporal precision fivefold compared to direct timing resolution of calcium signal. PMID:23711821

  15. Activation of Notch-Mediated Protective Signaling in the Myocardium

    PubMed Central

    Gude, Natalie A.; Emmanuel, Gregory; Wu, Weitao; Cottage, Christopher T.; Fischer, Kimberlee; Quijada, Pearl; Muraski, John A.; Alvarez, Roberto; Rubio, Marta; Schaefer, Eric; Sussman, Mark A.

    2013-01-01

    The Notch network regulates multiple cellular processes, including cell fate determination, development, differentiation, proliferation, apoptosis, and regeneration. These processes are regulated via Notch-mediated activity that involves hepatocyte growth factor (HGF)/c-Met receptor and phosphatidylinositol 3-kinase/Akt signaling cascades. The impact of HGF on Notch signaling was assessed following myocardial infarction as well as in cultured cardiomyocytes. Notch1 is activated in border zone cardiomyocytes coincident with nuclear c-Met following infarction. Intramyocardial injection of HGF enhances Notch1 and Akt activation in adult mouse myocardium. Corroborating evidence in cultured cardiomyocytes shows treatment with HGF or insulin increases levels of Notch effector Hes1 in immunoblots, whereas overexpression of activated Notch intracellular domain prompts a 3-fold increase in phosphorylated Akt. Infarcted hearts injected with adenoviral vector expressing Notch intracellular domain treatment exhibit improved hemodynamic function in comparison with control mice after 4 weeks, implicating Notch signaling in a cardioprotective role following cardiac injury. These results indicate Notch activation in cardiomyocytes is mediated through c-Met and Akt survival signaling pathways, and Notch1 signaling in turn enhances Akt activity. This mutually supportive crosstalk suggests a positive survival feedback mechanism between Notch and Akt signaling in adult myocardium following injury. PMID:18369158

  16. Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding Ras and activating Ras signaling.

    PubMed

    Song, Shumei; Ji, Baoan; Ramachandran, Vijaya; Wang, Huamin; Hafley, Margarete; Logsdon, Craig; Bresalier, Robert S

    2012-01-01

    Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease. PMID:22900040

  17. Signal peptides are allosteric activators of the protein translocase.

    PubMed

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G; Economou, Anastassios

    2009-11-19

    Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping' that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  18. N-myc downstream regulated gene 2 overexpression reduces matrix metalloproteinase-2 and -9 activities and cell invasion of A549 lung cancer cell line in vitro

    PubMed Central

    Faraji, Seyed Nooredin; Mojtahedi, Zahra; Ghalamfarsa, Ghasem; Takhshid, Mohammad Ali

    2015-01-01

    Objective(s): N-myc downstream regulated gene 2 (NDRG2) is a candidate gene for tumor suppression. The expression of NDRG2 is down-regulated in several tumors including lung cancer. The aim of this study was to explore the effect of NDRG2 overexpression on invasion, migration, and enzymatic activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in human lung adenocarcinoma A549 cells. Materials and Methods: A recombinant plasmid encoding green fluorescent protein (GFP)-tagged NDRG2 (pCMV6-AC-NDRG2-GFP) was used to overexpress GFP-tagged NDRG2 in A549 cells. The cells in the experimental group and those in the control group were transfected with pCMV6-AC-NDRG2-GFP and a control plasmid without NDRG2 (pCMV6-AC-GFP), respectively. Fluorescent microscopy and flowcytometry analysis of GFP expression were used to evaluate the cellular expression of GFP-tagged NDRG2 and the efficiency of transfection. The effects of NDRG2 expression on cell invasion and migration were evaluated using transwell filter migration assay. The gelatinase activity of secreted MMP-2 and MMP-9 was measured by gelatin zymography. Results: Our results demonstrated the expression of GFP-tagged NDRG2 in the cytoplasm and nucleus of A549 cells. The findings of transwell assay showed that NDRG2 overexpression reduced migration and invasion of A549 cells compared to control cells. Gelatin zymography analyses revealed that NDRG2 overexpression decreased the gelatinase activity of secreted MMP-2 and MMP-9. Conclusion: These findings suggest that NDRG2 may be a new anti-invasion factor in lung cancer that inhibits MMPs activities. PMID:26557966

  19. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor1[OPEN

    PubMed Central

    Kim, Mi Jung

    2016-01-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15. However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. PMID:27246098

  20. A historical perspective on the lateral diffusion model of GTPase activation and related coupling of membrane signaling proteins

    PubMed Central

    Liebman, Paul A

    2014-01-01

    Aspects of our discovery of lateral diffusion of the G protein coupled receptor (GPCR) rhodopsin and that a single activated rhodopsin can non-covalently catalyze GTP binding to thousands of GTPases per second on rod disk membranes via this diffusion are summarized herein. Rapid GTPase coupling to membrane-bound phosphodiesterase (PDE) further amplifies the signal via cGMP hydrolysis, essential to visual transduction. Important generalizations from this work are that biomembranes can uniquely concentrate, orient for reaction and provide a solvent appropriate to rapid, powerful and appropriately controlled sequential interaction of signaling proteins. Of equal importance to function is timely control and termination of such powerful amplification via receptor phosphorylation (quenching) and arrestin binding. Downstream kinetic modulation by GTPase activating proteins (GAPs) and regulators of G protein signaling (RGS) and related mechanisms as well as limitations set by membrane domain fencing, structural protein binding etc. can be essential in relevant systems. PMID:25279248

  1. Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    PubMed Central

    Fritsch, Cornelia; Klaver, Arjen; Kanger, Johannes S.; Jovin, Thomas M.; Arndt-Jovin, Donna J.

    2013-01-01

    Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor

  2. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

    PubMed Central

    Juo, P; Kuo, C J; Reynolds, S E; Konz, R F; Raingeaud, J; Davis, R J; Biemann, H P; Blenis, J

    1997-01-01

    The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades. PMID:8972182

  3. Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

    PubMed Central

    Sangphech, Naunpun; Osborne, Barbara A.; Palaga, Tanapat

    2014-01-01

    Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phosphoproteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via Gαi/Gαq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle

  4. FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

    PubMed

    Kamei, Yasutomi; Hattori, Maki; Hatazawa, Yukino; Kasahara, Tomomi; Kanou, Masanobu; Kanai, Sayaka; Yuan, Xunmei; Suganami, Takayoshi; Lamers, Wouter H; Kitamura, Tadahiro; Ogawa, Yoshihiro

    2014-09-15

    Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle. PMID:25074987

  5. In Vivo Mechanical Loading Rapidly Activates β–catenin Signaling in Osteocytes through a Prostaglandin Mediated Mechanism

    PubMed Central

    Lara-Castillo, N; Kim-Weroha, NA; Kamel, MA; Javaheri, B; Ellies, DL; Krumlauf, RE; Thiagarajan, G; Johnson, ML

    2015-01-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in the in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1 hour post loading, spreading to additional osteocytes and finally to cells on the bone surface by 24 hrs. This early activation at 1 hour appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly down regulated in osteocytes in vivo at 24 hours post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo. PMID:25836764

  6. In vivo mechanical loading rapidly activates β-catenin signaling in osteocytes through a prostaglandin mediated mechanism.

    PubMed

    Lara-Castillo, N; Kim-Weroha, N A; Kamel, M A; Javaheri, B; Ellies, D L; Krumlauf, R E; Thiagarajan, G; Johnson, M L

    2015-07-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24h. This early activation at 1h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo. PMID:25836764

  7. Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration.

    PubMed

    Han, Pingping; Ivanovski, Saso; Crawford, Ross; Xiao, Yin

    2015-07-01

    Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β-catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well-organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs. PMID:25556853

  8. Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity

    PubMed Central

    Baba, Shiro; Onga, Kazuko; Kakizawa, Sho; Ohyama, Kyoji; Yasuda, Kunihiko; Otsubo, Hiroshi; Scott, Brian W.; Burnham, W. McIntyre; Matsuo, Takayuki; Nagata, Izumi; Mori, Nozomu

    2016-01-01

    BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy. PMID:27273072

  9. Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity.

    PubMed

    Baba, Shiro; Onga, Kazuko; Kakizawa, Sho; Ohyama, Kyoji; Yasuda, Kunihiko; Otsubo, Hiroshi; Scott, Brian W; Burnham, W McIntyre; Matsuo, Takayuki; Nagata, Izumi; Mori, Nozomu

    2016-01-01

    BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy. PMID:27273072

  10. Coco is a dual activity modulator of TGFβ signaling

    PubMed Central

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  11. View downstream of timber guide wall downstream from SE corner ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View downstream of timber guide wall downstream from SE corner of lock, view towards east - St. Lucie Canal, St. Lucie Lock No. 1, St. Lucie, Cross State Canal, Okeechobee Intracoastal Waterway, Stuart, Martin County, FL

  12. Mechanistic and Signaling Analysis of Muc4-ErbB2 Signaling Module: New Insights into the Mechanism of Ligand-Independent ErbB2 Activity

    PubMed Central

    Kozloski, Goldi A.; Carothers Carraway, Coralie A.; Carraway, Kermit L.

    2010-01-01

    SUMMARY The membrane mucin Muc4 is aberrantly expressed in numerous epithelial carcinomas and is currently used as a cancer diagnostic and prognostic tool. Muc4 can also potentiate signal transduction by modulating differential ErbB2 phosphorylation in the absence and in the presence of the ErbB3 soluble ligand heregulin (HRG-β1). These features of Muc4 suggest that Muc4 is not merely a cancer marker, but an oncogenic factor with a unique binding/activation relationship with the receptor ErbB2. In the present study, we examined the signaling mechanisms that are associated with the Muc4-ErbB2 module by analyzing ErbB2 differential signaling in response to Muc4 expression. Our study was carried out in the A375 human melanoma and BT-474 breast cancer cell lines as our model systems. Quantitative and comparative signaling modulations were evaluated by immunoblot using phospho-specific antibodies, and densitometry analysis. Signaling complex components were identified by chemical cross-linking, fractionation by gel filtration, immunoprecipitation and immunoblotting. Activated downstream signaling pathways were analyzed by an antibody microarray screen and immunoblot analyses. Our results indicate that Muc4 modulates ErbB2 signaling potential significantly by stabilizing and directly interacting with the ErbB2-ErbB3 heterodimer. Further analyses indicate that Muc4 promotes ErbB2 autocatalysis, but it has no effect on ErbB3 phosphorylation, although the chemical cross linking data indicated that the signaling module is composed of Muc4, ErbB2 and ErbB3. Our microarray analysis indicates that Muc4 expression promotes cell migration by increasing the phosphorylation of the focal adhesion kinase (FAK) and also through an increase in the levels of β-catenin. PMID:20432461

  13. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity

    PubMed Central

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-01-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important. PMID:25077176

  14. Ciclopirox olamine inhibits mTORC1 signaling by activation of AMPK.

    PubMed

    Zhou, Hongyu; Shang, Chaowei; Wang, Min; Shen, Tao; Kong, Lingmei; Yu, Chunlei; Ye, Zhennan; Luo, Yan; Liu, Lei; Li, Yan; Huang, Shile

    2016-09-15

    Ciclopirox olamine (CPX), an off-patent antifungal agent, has recently been identified as a potential anticancer agent. The mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation and survival. Little is known about whether and how CPX executes its anticancer action by inhibiting mTOR. Here we show that CPX inhibited the phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), two downstream effector molecules of mTOR complex 1 (mTORC1), in a spectrum of human tumor cells, indicating that CPX inhibits mTORC1 signaling. Using rhabdomyosarcoma cells as an experimental model, we found that expression of constitutively active mTOR (E2419K) conferred resistance to CPX inhibition of cell proliferation, suggesting that CPX inhibition of mTORC1 contributed to its anticancer effect. In line with this, treatment with CPX inhibited tumor growth and concurrently suppressed mTORC1 signaling in RD xenografts. Mechanistically, CPX inhibition of mTORC1 was neither via inhibition of IGF-I receptor or phosphoinositide 3-kinase (PI3K), nor by activation of phosphatase and tensin homolog (PTEN). Instead, CPX inhibition of mTORC1 was attributed to activation of AMP-activated protein kinase (AMPK)-tuberous sclerosis complexes (TSC)/raptor pathways. This is supported by the findings that CPX activated AMPK; inhibition of AMPK with Compound C or ectopic expression of dominant negative AMPKα partially prevented CPX from inhibiting mTORC1; silencing TSC2 attenuated CPX inhibition of mTORC1; and CPX also increased AMPK-mediated phosphorylation of raptor (S792). Therefore, the results indicate that CPX exerts the anticancer effect by activating AMPK, resulting in inhibition of mTORC1 signaling. PMID:27396756

  15. Pharmacology of Signaling Induced by Dopamine D1-Like Receptor Activation

    PubMed Central

    Undieh, Ashiwel S.

    2010-01-01

    Dopamine D1-like receptors consisting of D1 and D5 subtypes are intimately implicated in dopaminergic regulation of fundamental neurophysiologic processes such as mood, motivation, cognitive function, and motor activity. Upon stimulation, D1-like receptors initiate signal transduction cascades that are mediated through adenylyl cyclase or phosphoinositide metabolism, with subsequent enhancement of multiple downstream kinase cascades. The latter actions propagate and further amplify the receptor signals, thus predisposing D1-like receptors to multifaceted interactions with various other mediators and receptor systems. The adenylyl cyclase response to dopamine or selective D1-like receptor agonists is reliably associated with the D1 subtype, while emerging evidence indicates that the phosphoinositide responses in native brain tissues may be preferentially mediated through stimulation of the D5 receptor. Besides classic coupling of each receptor subtype to specific G proteins, additional biophysical models are advanced in attempts to account for differential subcellular distribution, heteromolecular oligomerization, and activity-dependent selectivity of the receptors. It is expected that significant advances in understanding of dopamine neurobiology will emerge from current and anticipated studies directed at uncovering the molecular mechanisms of D5 coupling to phosphoinositide signaling, the structural features that might enhance pharmacological selectivity for D5 versus D1 subtypes, the mechanism by which dopamine may modulate phosphoinositide synthesis, the contributions of the various responsive signal mediators to D1 or D5 interactions with D2-like receptors, and the spectrum of dopaminergic functions that may be attributed to each receptor subtype and signaling pathway. PMID:20547182

  16. X-ray irradiation activates K+ channels via H2O2 signaling.

    PubMed

    Gibhardt, Christine S; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  17. X-ray irradiation activates K+ channels via H2O2 signaling

    PubMed Central

    Gibhardt, Christine S.; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  18. Activation of cell signaling via optical manipulation of gold-coated liposomes encapsulating signaling molecules

    NASA Astrophysics Data System (ADS)

    Orsinger, Gabriel V.; Leung, Sarah J.; Romanowski, Marek

    2013-02-01

    Many diseases involve changes in cell signaling cascades, as seen commonly in drug resistant cancers. To better understand these intricate signaling events in diseased cells and tissues, experimental methods of probing cellular communication at a single to multi-cell level are required. We recently introduced a general platform for activation of selected signaling pathways by optically controlled delivery and release of water soluble factors using gold-coated liposomes. In the example presented here, we encapsulated inositol trisphosphate (IP3), a ubiquitous intracellular secondary messenger involved in GPCR and Akt signaling cascades, within 100 nm gold-coated liposomes. The high polarizability of the liposome's unique gold pseudo-shell allows stable optical trapping for subcellular manipulation in the presence of cells. We take this optical manipulation further by optically injecting IP3-containing liposomes into the cytosol of a single cell to initiate localized cell signaling. Upon optical injection of liposomal IP3 into a single ovarian carcinoma cell, we observed localized activation as reported by changes in Indo-1 fluorescence intensity. With established gap junctions between the injected cell and neighboring cells, we monitored propagation of this signaling to and through nearby cells.

  19. Correlation of nighttime MF signal strength with solar activity

    NASA Astrophysics Data System (ADS)

    Kohata, Hiroki; Kimura, Iwane; Wakai, Noboru; Ogawa, Tadahiko

    Observations of the signal strength of MF broadcasting signals (774/770 kHz) transmitted from Akita, Japan, on board the Japanese Antarctic ice breaker Fuji, bound from Japan to Syowa station, Antarctica, have revealed an interesting positive correlation between strengths of long distance signals propagating at night and solar activity. It is already known that MF propagation characteristics in North America show a negative correlation with solar activity. The present paper, interprets the results by using the multihop method with full-wave analysis. The difference in correlation with solar activity between the results of Fuji and those in North America can be elucidated if it is assumed that there is a ledge in the electron-density profile around an altitude range of 85 to 90 km and that the density of the ledge is smaller in the North American region than in the equatorial region.

  20. Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway

    PubMed Central

    Liu, Hong-Shuai; Shi, Hai-Lian; Huang, Fei; Peterson, Karin E.; Wu, Hui; Lan, Yun-Yi; Zhang, Bei-Bei; He, Yi-Xin; Woods, Tyson; Du, Min; Wu, Xiao-Jun; Wang, Zheng-Tao

    2016-01-01

    Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases. PMID:26750705

  1. Combined genetic and transcriptomic analysis reveals three major signalling pathways activated by Myc-LCOs in Medicago truncatula.

    PubMed

    Camps, Céline; Jardinaud, Marie-Françoise; Rengel, David; Carrère, Sébastien; Hervé, Christine; Debellé, Frédéric; Gamas, Pascal; Bensmihen, Sandra; Gough, Clare

    2015-10-01

    Myc-LCOs are newly identified symbiotic signals produced by arbuscular mycorrhizal (AM) fungi. Like rhizobial Nod factors, they are lipo-chitooligosaccharides that activate the common symbiotic signalling pathway (CSSP) in plants. To increase our limited understanding of the roles of Myc-LCOs we aimed to analyse Myc-LCO-induced transcriptional changes and their genetic control. Whole genome RNA sequencing (RNA-seq) was performed on roots of Medicago truncatula wild-type plants, and dmi3 and nsp1 symbiotic mutants affected in nodulation and mycorrhizal signalling. Plants were treated separately with the two major types of Myc-LCOs, sulphated and nonsulphated. Generalized linear model analysis identified 2201 differentially expressed genes and classified them according to genotype and/or treatment effects. Three genetic pathways for Myc-LCO-regulation of transcriptomic reprogramming were highlighted: DMI3- and NSP1-dependent; DMI3-dependent and NSP1-independent; and DMI3- and NSP1-independent. Comprehensive analysis revealed overlaps with previous AM studies, and highlighted certain functions, especially signalling components and transcription factors. These data provide new insights into mycorrhizal signalling mechanisms, supporting a role for NSP1, and specialisation for NSP1-dependent and -independent pathways downstream of DMI3. Our data also indicate significant Myc-LCO-activated signalling upstream of DMI3 and/or parallel to the CSSP and some constitutive activity of the CSSP. PMID:25919491

  2. Aberrant Activation of Notch Signaling Inhibits PROX1 Activity to Enhance the Malignant Behavior of Thyroid Cancer Cells.

    PubMed

    Choi, Dongwon; Ramu, Swapnika; Park, Eunkyung; Jung, Eunson; Yang, Sara; Jung, Wonhyeuk; Choi, Inho; Lee, Sunju; Kim, Kyu Eui; Seong, Young Jin; Hong, Mingu; Daghlian, George; Kim, Daniel; Shin, Eugene; Seo, Jung In; Khatchadourian, Vicken; Zou, Mengchen; Li, Wei; De Filippo, Roger; Kokorowski, Paul; Chang, Andy; Kim, Steve; Bertoni, Ana; Furlanetto, Tania Weber; Shin, Sung; Li, Meng; Chen, Yibu; Wong, Alex; Koh, Chester; Geliebter, Jan; Hong, Young-Kwon

    2016-02-01

    Papillary thyroid cancer (PTC) is one of the most common endocrine malignancies associated with significant morbidity and mortality. Although multiple studies have contributed to a better understanding of the genetic alterations underlying this frequently arising disease, the downstream molecular effectors that impact PTC pathogenesis remain to be further defined. Here, we report that the regulator of cell fate specification, PROX1, becomes inactivated in PTC through mRNA downregulation and cytoplasmic mislocalization. Expression studies in clinical specimens revealed that aberrantly activated NOTCH signaling promoted PROX1 downregulation and that cytoplasmic mislocalization significantly altered PROX1 protein stability. Importantly, restoration of PROX1 activity in thyroid carcinoma cells revealed that PROX1 not only enhanced Wnt/β-catenin signaling but also regulated several genes known to be associated with PTC, including thyroid cancer protein (TC)-1, SERPINA1, and FABP4. Furthermore, PROX1 reexpression suppressed the malignant phenotypes of thyroid carcinoma cells, such as proliferation, motility, adhesion, invasion, anchorage-independent growth, and polyploidy. Moreover, animal xenograft studies demonstrated that restoration of PROX1 severely impeded tumor formation and suppressed the invasiveness and the nuclear/cytoplasmic ratio of PTC cells. Taken together, our findings demonstrate that NOTCH-induced PROX1 inactivation significantly promotes the malignant behavior of thyroid carcinoma and suggest that PROX1 reactivation may represent a potential therapeutic strategy to attenuate disease progression. PMID:26609053

  3. Notch signaling promotes osteoclast maturation and resorptive activity.

    PubMed

    Ashley, Jason W; Ahn, Jaimo; Hankenson, Kurt D

    2015-11-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors. PMID:25914241

  4. Aberrant activation of Wnt/β-catenin signaling pathway contributes to the sequential progression of DMBA-induced HBP carcinomas.

    PubMed

    Vidya Priyadarsini, Ramamurthi; Senthil Murugan, Ramalingam; Nagini, Siddavaram

    2012-01-01

    Wnt signaling pathway mediated via interactions between β-catenin and members of the TCF/LEF-1 family of transcription factors plays a central role in the regulation of epithelial cell proliferation, apoptosis, differentiation, adhesion, epithelial-mesenchymal transition, and invasion. Aberrant activation of the Wnt/β-catenin signaling pathway with overexpression of Wnt and Fz, mutations of APC, β-catenin, and axin 1, and cytoplasmic accumulation of β-catenin have been frequently reported in a broad spectrum of human malignancies including oral squamous cell carcinomas (OSCCs). However, changes in the components of the Wnt signaling pathway have not been documented during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis, a paradigm for oral oncogenesis and chemointervention. In this study, we evaluated the role of β-catenin accumulation and Wnt ligands, Wnt signaling members (Fz, Dvl, APC, GSK-3β, axin, and WIF) and the downstream targets of Wnt (cyclin D1, MMP-2, and MMP-9) during the sequential progression of DMBA-induced HBP carcinomas by semi-quantitative RT-PCR and western blot analyses. Our data reveal a correlation between β-catenin accumulation and activation of Wnt signaling, and its downstream effector molecules during the sequential development of HBP carcinomas from hyperplasia to invasive carcinoma through dysplasia. Our data also support a pivotal role for β-catenin in the malignant transition of the HBP. Aberrant Wnt signaling may be a hallmark of progression to malignancy during DMBA-induced HBP carcinogenesis and could be a potential preventive and therapeutic target for suppression of OSCC. PMID:21924667

  5. Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.

    PubMed

    Wang, Ronghua; Sun, Qian; Wang, Peng; Liu, Man; Xiong, Si; Luo, Jing; Huang, Hai; Du, Qiang; Geller, David A; Cheng, Bin

    2016-02-01

    Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC. PMID:26735577

  6. A Temporal Window for Signal Activation Dictates the Dimensions of a Nodal Signaling Domain

    PubMed Central

    van Boxtel, Antonius L.; Chesebro, John E.; Heliot, Claire; Ramel, Marie-Christine; Stone, Richard K.; Hill, Caroline S.

    2015-01-01

    Summary Morphogen signaling is critical for the growth and patterning of tissues in embryos and adults, but how morphogen signaling gradients are generated in tissues remains controversial. The morphogen Nodal was proposed to form a long-range signaling gradient via a reaction-diffusion system, on the basis of differential diffusion rates of Nodal and its antagonist Lefty. Here we use a specific zebrafish Nodal biosensor combined with immunofluorescence for phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse over a long range. Instead, short-range Nodal signaling activation in a temporal window is sufficient to determine the dimensions of the Nodal signaling domain. The size of this temporal window is set by the differentially timed production of Nodal and Lefty, which arises mainly from repression of Lefty translation by the microRNA miR-430. Thus, temporal information is transformed into spatial information to define the dimensions of the Nodal signaling domain and, consequently, to specify mesendoderm. PMID:26506307

  7. A Temporal Window for Signal Activation Dictates the Dimensions of a Nodal Signaling Domain.

    PubMed

    van Boxtel, Antonius L; Chesebro, John E; Heliot, Claire; Ramel, Marie-Christine; Stone, Richard K; Hill, Caroline S

    2015-10-26

    Morphogen signaling is critical for the growth and patterning of tissues in embryos and adults, but how morphogen signaling gradients are generated in tissues remains controversial. The morphogen Nodal was proposed to form a long-range signaling gradient via a reaction-diffusion system, on the basis of differential diffusion rates of Nodal and its antagonist Lefty. Here we use a specific zebrafish Nodal biosensor combined with immunofluorescence for phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse over a long range. Instead, short-range Nodal signaling activation in a temporal window is sufficient to determine the dimensions of the Nodal signaling domain. The size of this temporal window is set by the differentially timed production of Nodal and Lefty, which arises mainly from repression of Lefty translation by the microRNA miR-430. Thus, temporal information is transformed into spatial information to define the dimensions of the Nodal signaling domain and, consequently, to specify mesendoderm. PMID:26506307

  8. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling.

    PubMed

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  9. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling

    PubMed Central

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  10. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation.

    PubMed

    Phong, Binh L; Avery, Lyndsay; Sumpter, Tina L; Gorman, Jacob V; Watkins, Simon C; Colgan, John D; Kane, Lawrence P

    2015-12-14

    T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation. PMID:26598760

  11. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation

    PubMed Central

    Phong, Binh L.; Avery, Lyndsay; Sumpter, Tina L.; Gorman, Jacob V.; Watkins, Simon C.; Colgan, John D.

    2015-01-01

    T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation. PMID:26598760

  12. Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: a computational study.

    PubMed

    Ganguli, Piyali; Chowdhury, Saikat; Bhowmick, Rupa; Sarkar, Ram Rup

    2015-10-01

    Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in these co-receptor signalling pathways may lead to an altered expression pattern of the effector molecules. To study the propagation of such defects with time and their effect on the intracellular protein expression patterns, a comprehensive and largest pathway map of T-cell activation network is reconstructed manually. The entire pathway reactions are then translated using logical equations and simulated using the published time series microarray expression data as inputs. After validating the model, the effect of in silico knock down of co-receptor molecules on the expression patterns of their downstream proteins is studied and simultaneously the changes in the phenotypic behaviours of the T-cell population are predicted, which shows significant variations among the proteins expression and the signalling routes through which the response is propagated in the cytoplasm. This integrative computational approach serves as a valuable technique to study the changes in protein expression patterns and helps to predict variations in the cellular behaviour. PMID:26564978

  13. CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling.

    PubMed

    Rodríguez-Nieves, José A; Patalano, Susan C; Almanza, Diego; Gharaee-Kermani, Mehrnaz; Macoska, Jill A

    2016-01-01

    Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis. PMID:27434301

  14. CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling

    PubMed Central

    Rodríguez-Nieves, José A.; Patalano, Susan C.; Almanza, Diego; Gharaee-Kermani, Mehrnaz; Macoska, Jill A.

    2016-01-01

    Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis. PMID:27434301

  15. Metabolic signals and innate immune activation in obesity and exercise.

    PubMed

    Ringseis, Robert; Eder, Klaus; Mooren, Frank C; Krüger, Karsten

    2015-01-01

    The combination of a sedentary lifestyle and excess energy intake has led to an increased prevalence of obesity which constitutes a major risk factor for several co-morbidities including type 2 diabetes and cardiovascular diseases. Intensive research during the last two decades has revealed that a characteristic feature of obesity linking it to insulin resistance is the presence of chronic low-grade inflammation being indicative of activation of the innate immune system. Recent evidence suggests that activation of the innate immune system in the course of obesity is mediated by metabolic signals, such as free fatty acids (FFAs), being elevated in many obese subjects, through activation of pattern recognition receptors thereby leading to stimulation of critical inflammatory signaling cascades, like IκBα kinase/nuclear factor-κB (IKK/NF- κB), endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) and NOD-like receptor P3 (NLRP3) inflammasome pathway, that interfere with insulin signaling. Exercise is one of the main prescribed interventions in obesity management improving insulin sensitivity and reducing obesity- induced chronic inflammation. This review summarizes current knowledge of the cellular recognition mechanisms for FFAs, the inflammatory signaling pathways triggered by excess FFAs in obesity and the counteractive effects of both acute and chronic exercise on obesity-induced activation of inflammatory signaling pathways. A deeper understanding of the effects of exercise on inflammatory signaling pathways in obesity is useful to optimize preventive and therapeutic strategies to combat the increasing incidence of obesity and its comorbidities. PMID:25825956

  16. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  17. Imaging the coordination of multiple signaling activities in living cells

    PubMed Central

    Welch, Christopher M.; Elliott, Hunter; Danuser, Gaudenz; Hahn, Klaus M.

    2013-01-01

    Preface Cellular signal transduction occurs in complex and redundant interaction networks that are best examined at the level of single cells by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches to determine the relationships between different networks, studied in separate cells. PMID:22016058

  18. TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

    PubMed

    Lehnert, Corinna; Weiswange, Maxi; Jeremias, Irmela; Bayer, Carina; Grunert, Michaela; Debatin, Klaus-Michael; Strauss, Gudrun

    2014-10-15

    The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. PMID:25217163

  19. Hyperpolarization-activated ion channels as targets for nitric oxide signalling in deep cerebellar nuclei

    PubMed Central

    Wilson, Gary W; Garthwaite, John

    2010-01-01

    Most biological effects of nitric oxide (NO) in the brain are mediated by guanylyl cyclase-coupled NO receptors, whose activation results in increased intracellular cGMP levels. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels, which bind cGMP or cAMP directly, were recently suggested to be a target. The aim here was to test this possibility more directly. Neurones of the rat deep cerebellar nuclei were selected for this purpose, their suitability being attested by immunocytochemistry showing that the principal neurones expressed guanylyl cyclase protein and that NO synthase-containing fibres were abundant in the neuropil. Using whole-cell voltage-clamp recording, HCN channels in the neurones were activated in response to isoprenaline and exogenous cAMP but only occasionally did they respond to NO, although exogenous cGMP was routinely effective. With the less invasive sharp microelectrode recording technique, however, exogenous NO modulated the channels reproducibly, as measured by the size of the HCN channel-mediated voltage sag following hyperpolarization. Moreover, NO also blunted the subsequent rebound depolarizing potentials, consistent with it increasing the hyperpolarization-activated current. Optimizing the whole-cell solution to improve the functioning of NO-activated guanylyl cyclase failed to restore NO sensitivity. Minimizing cellular dialysis by using the perforated-patch technique, however, was successful. The results provide evidence that HCN channels are potential downstream mediators of NO signalling in deep cerebellar nuclei neurones and suggest that the more general importance of this transduction pathway may have been overlooked previously because of unsuitable recording methods. PMID:20529121

  20. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  1. Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells

    PubMed Central

    Lee, Hye-Jung; Bae, Song Yi; Jung, Cholomi; Park, Hyen Joo; Lee, Sang Kook

    2015-01-01

    Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2-mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of p-AMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization. PMID:26656173

  2. Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation

    PubMed Central

    Ribeiro, Sérgio T.; Ribot, Julie C.; Silva-Santos, Bruno

    2015-01-01

    The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy. PMID:25674089

  3. Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells

    SciTech Connect

    Chang, C.-H.; Yu, F.-Y.; Wang, L.-T.; Lin, Y.-S.; Liu, B.-H.

    2009-06-15

    Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reporter assays. Bioinformatic analyses indicated two genes Gadd45{beta} and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45{beta} mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.

  4. Activation of B cells by non-canonical helper signals

    PubMed Central

    Cerutti, Andrea; Cols, Montserrat; Puga, Irene

    2012-01-01

    Cognate interaction between T and B lymphocytes of the adaptive immune system is essential for the production of high-affinity antibodies against microbes, and for the establishment of long-term immunological memory. Growing evidence shows that—in addition to presenting antigens to T and B cells—macrophages, dendritic cells and other cells of the innate immune system provide activating signals to B cells, as well as survival signals to antibody-secreting plasma cells. Here, we discuss how these innate immune cells contribute to the induction of highly diversified and temporally sustained antibody responses, both systemically and at mucosal sites of antigen entry. PMID:22868664

  5. Molecular hydrogen suppresses activated Wnt/β-catenin signaling.

    PubMed

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  6. Molecular hydrogen suppresses activated Wnt/β-catenin signaling

    PubMed Central

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  7. G Protein Activation Stimulates Phospholipase D Signaling in Plants.

    PubMed Central

    Munnik, T.; Arisz, S. A.; De Vrije, T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals. PMID:12242371

  8. Chemical activation of a food deprivation signal extends lifespan.

    PubMed

    Lucanic, Mark; Garrett, Theo; Yu, Ivan; Calahorro, Fernando; Asadi Shahmirzadi, Azar; Miller, Aaron; Gill, Matthew S; Hughes, Robert E; Holden-Dye, Lindy; Lithgow, Gordon J

    2016-10-01

    Model organisms subject to dietary restriction (DR) generally live longer. Accompanying this lifespan extension are improvements in overall health, based on multiple metrics. This indicates that pharmacological treatments that mimic the effects of DR could improve health in humans. To find new chemical structures that extend lifespan, we screened 30 000 synthetic, diverse drug-like chemicals in Caenorhabditis elegans and identified several structurally related compounds that acted through DR mechanisms. The most potent of these NP1 impinges upon a food perception pathway by promoting glutamate signaling in the pharynx. This results in the overriding of a GPCR pathway involved in the perception of food and which normally acts to decrease glutamate signals. Our results describe the activation of a dietary restriction response through the pharmacological masking of a novel sensory pathway that signals the presence of food. This suggests that primary sensory pathways may represent novel targets for human pharmacology. PMID:27220516

  9. Berbamine enhances the antineoplastic activity of gemcitabine in pancreatic cancer cells by activating transforming growth factor-β/Smad signaling.

    PubMed

    Jin, Xiaoli; Wu, Yulian

    2014-05-01

    Drug-resistance to gemcitabine chemotherapy in pancreatic cancer is still an unsolved problem. Combinations of other chemotherapy drugs with gemcitabine have been shown to increase the efficacy of gemcitabine-based treatment. In this study, the effect of berbamine on the antitumor activity of gemcitabine was evaluated in human pancreatic cancer cell lines Bxpc-3 and Panc-1, and the underlying mechanisms were explored. Our results demonstrated that berbamine exhibited a time- and dose-dependent inhibitory effect in the pancreatic cancer cell lines. Berbamine enhanced gemcitabine-induced cell growth inhibition and apoptosis in these cells. Combined treatment of berbamine and gemcitabine resulted in down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL) and up-regulation of pro-apoptotic proteins (Bax, Bid). More importantly, berbamine treatment in combination with gemcitabine activated the transforming growth factor-β/Smad (TGF-β/Smad) signaling pathway, as a result of a decrease in Smad7 and an increase in transforming growth factor-β receptor II (TβRII) expression. Changes in downstream targets of Smad7, such as up-regulation of p21 and down-regulation of c-Myc and Cyclin D1 were also observed. Therefore, berbamine could enhance the antitumor activity of gemcitabine by inhibiting cell growth and inducing apoptosis, possibly through the regulation of the expression of apoptosis-related proteins and the activation of TGF-β/Smad signaling pathway. Our study indicates that berbamine may be a promising candidate to be used in combination with gemcitabine for pancreatic cancer treatment. PMID:24619961

  10. Constitutive activation of MET signaling impairs myogenic differentiation of rhabdomyosarcoma and promotes its development and progression

    PubMed Central

    Szewczyk, Barbara; Adamus, Tomasz; Lukasiewicz, Ewa; Miekus, Katarzyna; Majka, Marcin

    2015-01-01

    Rhabdomyosarcoma (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression. PMID:26384300

  11. Constitutive activation of MET signaling impairs myogenic differentiation of rhabdomyosarcoma and promotes its development and progression.

    PubMed

    Skrzypek, Klaudia; Kusienicka, Anna; Szewczyk, Barbara; Adamus, Tomasz; Lukasiewicz, Ewa; Miekus, Katarzyna; Majka, Marcin

    2015-10-13

    Rhabdomyosarcoma (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression. PMID:26384300

  12. Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation.

    PubMed

    Sumit, M; Neubig, R R; Takayama, S; Linderman, J J

    2015-11-01

    Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways. PMID:26374065

  13. Robust Indoor Human Activity Recognition Using Wireless Signals.

    PubMed

    Wang, Yi; Jiang, Xinli; Cao, Rongyu; Wang, Xiyang

    2015-01-01

    Wireless signals-based activity detection and recognition technology may be complementary to the existing vision-based methods, especially under the circumstance of occlusions, viewpoint change, complex background, lighting condition change, and so on. This paper explores the properties of the channel state information (CSI) of Wi-Fi signals, and presents a robust indoor daily human activity recognition framework with only one pair of transmission points (TP) and access points (AP). First of all, some indoor human actions are selected as primitive actions forming a training set. Then, an online filtering method is designed to make actions' CSI curves smooth and allow them to contain enough pattern information. Each primitive action pattern can be segmented from the outliers of its multi-input multi-output (MIMO) signals by a proposed segmentation method. Lastly, in online activities recognition, by selecting proper features and Support Vector Machine (SVM) based multi-classification, activities constituted by primitive actions can be recognized insensitive to the locations, orientations, and speeds. PMID:26184231

  14. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia

    PubMed Central

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  15. Activation of a signaling cascade by cytoskeleton stretch.

    PubMed

    Tamada, Masako; Sheetz, Michael P; Sawada, Yasuhiro

    2004-11-01

    Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1. PMID:15525532

  16. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia.

    PubMed

    Ye, Qi; Jiang, Jue; Zhan, Guanqun; Yan, Wanyao; Huang, Liang; Hu, Yufeng; Su, Hexiu; Tong, Qingyi; Yue, Ming; Li, Hua; Yao, Guangmin; Zhang, Yonghui; Liu, Hudan

    2016-01-01

    Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML. PMID:27211848

  17. Analysis of signal degradation in an integrated active crossbar switch

    NASA Astrophysics Data System (ADS)

    Probst, David K.; Sodergren, Clifford C.; Krainak, Michael A.

    1996-01-01

    One of the most desirable features for a modern systems architecture is reconfigurability. It facilitates the sharing of various processing and memory resources among many different subsystems, thereby reducing the need for each subsystem to duplicate these resources. Reducing duplication also reduces size, weight, power consumption, and cost; all important considerations, especially in modern military systems. Sensor data requires very-wide- bandwidth, point-to-point connections, which are easily provided by fiber optics with good noise immunity, but the switching of such wide-bandwidth signals is problematic because electrical switches have both limited bandwidth and limited switching times. A wide- bandwidth, reconfigurable optical switch is required that overcomes coupling and splitting losses experienced by the optical signal passing through the switch so that sufficient signal- fidelity is maintained. In this paper, we investigate an integrated, active (i.e. amplifying) photonic crossbar switch to determine the signal degradation incurred for intensity modulation and direct detection using an NRZ data format and an ideal, matched-filter receiver. Various device configurations are analyzed in order to determine which produces the smallest degradation of the signal-to-noise ratio.

  18. FK866-induced NAMPT inhibition activates AMPK and downregulates mTOR signaling in hepatocarcinoma cells

    SciTech Connect

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Gebhardt, Rolf; Weiss, Thomas S.; Kiess, Wieland; Garten, Antje

    2015-03-06

    Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. - Highlights: • FK866 increases cell death in p53-deficient hepatocarcinoma cells. • AMPK/mTOR signaling is dysregulated in hepatocarcinoma cells. • FK866-induced NAMPT inhibition activates AMPK

  19. Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo.

    PubMed

    Block, Helena; Stadtmann, Anika; Riad, Daniel; Rossaint, Jan; Sohlbach, Charlotte; Germena, Giulia; Wu, Dianqing; Simon, Scott I; Ley, Klaus; Zarbock, Alexander

    2016-01-21

    Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest. PMID:26468229

  20. View of downstream debris field at the Merry Generator House, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of downstream debris field at the Merry Generator House, showing possible concrete generator seats, looking south - Arthur Holmes Merry Generator House, Signal Lake North of Range Road, Fort Gordon, Richmond County, GA

  1. Extracellular PKM2 induces cancer proliferation by activating the EGFR signaling pathway

    PubMed Central

    Hsu, Ming-Chuan; Hung, Wen-Chun; Yamaguchi, Hirohito; Lim, Seung-Oe; Liao, Hsin-Wei; Tsai, Chia-Hua; Hung, Mien-Chie

    2016-01-01

    Pyruvate kinase is a key enzyme in the glycolytic pathway that converts phosphoenolpyruvate to pyruvate, and the M2 isoform of pyruvate kinase (PKM2) is associated with cancer. PKM2 has been reported to function independently of its pyruvate kinase activity, which is crucial for cancer cell proliferation. Moreover, there is growing evidence indicating that dimeric PKM2 is released from tumor cells into the circulation of cancer patients. However, the role of secreted PKM2 in cancer is not well understood. Here, we found that the phosphorylation level of epidermal growth factor receptor (EGFR) significantly increased upon the exposure of cells to the recombinant PKM2 protein. In addition, secreted PKM2 induces EGFR phosphorylation and activates the EGFR downstream signaling in triple-negative breast cancer cells. In contrast, knocking down PKM2 decreased EGFR phosphorylation. Moreover, expression of R399E mutant PKM2, which has been reported to preferentially form a dimer, enhanced EGFR phosphorylation, cellular transformation, and cell proliferation more strongly than the wild-type PKM2. Thus, our study revealed a novel function of extracellular PKM2 in the promoting cancer cell proliferation through EGFR activation. PMID:27152240

  2. Inhibition of TGFbeta1 Signaling Attenutates ATM Activity inResponse to Genotoxic Stress

    SciTech Connect

    Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose; Ravani, Shraddha A.; Glick, Adam B.; Lavin, Martin J.; Koslov, Sergei; Shiloh, Yosef; Barcellos-Hoff, Mary Helen

    2006-09-15

    Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta}1 (TGF{beta}), which is activated by radiation, is a potent and pleiotropic mediator of physiological and pathological processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}1 null murine epithelial cells or human epithelial cells treated with a small molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17 and p53, reduced {gamma}H2AX radiation-induced foci, and increased radiosensitivity compared to TGF{beta} competent cells. We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM that directs epithelial cell stress responses, cell fate and tissue integrity. Thus, TGF{beta}1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.

  3. Systematic identification of signal-activated stochastic gene regulation.

    PubMed

    Neuert, Gregor; Munsky, Brian; Tan, Rui Zhen; Teytelman, Leonid; Khammash, Mustafa; van Oudenaarden, Alexander

    2013-02-01

    Although much has been done to elucidate the biochemistry of signal transduction and gene regulatory pathways, it remains difficult to understand or predict quantitative responses. We integrate single-cell experiments with stochastic analyses, to identify predictive models of transcriptional dynamics for the osmotic stress response pathway in Saccharomyces cerevisiae. We generate models with varying complexity and use parameter estimation and cross-validation analyses to select the most predictive model. This model yields insight into several dynamical features, including multistep regulation and switchlike activation for several osmosensitive genes. Furthermore, the model correctly predicts the transcriptional dynamics of cells in response to different environmental and genetic perturbations. Because our approach is general, it should facilitate a predictive understanding for signal-activated transcription of other genes in other pathways or organisms. PMID:23372015

  4. Gq signaling causes glomerular injury by activating TRPC6

    PubMed Central

    Wang, Liming; Jirka, Grant; Rosenberg, Paul B.; Buckley, Anne F.; Gomez, Jose A.; Fields, Timothy A.; Winn, Michelle P.; Spurney, Robert F.

    2015-01-01

    Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (GqQ209L, referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases. PMID:25844902

  5. Notch signals positively regulate activity of the mTOR pathway in T-cell acute lymphoblastic leukemia

    PubMed Central

    Chan, Steven M.; Weng, Andrew P.; Tibshirani, Robert; Aster, Jon C.

    2007-01-01

    Constitutive Notch activation is required for the proliferation of a subgroup of T-cell acute lymphoblastic leukemia (T-ALL). Downstream pathways that transmit pro-oncogenic signals are not well characterized. To identify these pathways, protein microarrays were used to profile the phosphorylation state of 108 epitopes on 82 distinct signaling proteins in a panel of 13 T-cell leukemia cell lines treated with a gamma-secretase inhibitor (GSI) to inhibit Notch signals. The microarray screen detected GSI-induced hypophosphorylation of multiple signaling proteins in the mTOR pathway. This effect was rescued by expression of the intracellular domain of Notch and mimicked by dominant negative MAML1, confirming Notch specificity. Withdrawal of Notch signals prevented stimulation of the mTOR pathway by mitogenic factors. These findings collectively suggest that the mTOR pathway is positively regulated by Notch in T-ALL cells. The effect of GSI on the mTOR pathway was independent of changes in phosphatidylinositol-3 kinase and Akt activity, but was rescued by expression of c-Myc, a direct transcriptional target of Notch, implicating c-Myc as an intermediary between Notch and mTOR. T-ALL cell growth was suppressed in a highly synergistic manner by simultaneous treatment with the mTOR inhibitor rapamycin and GSI, which represents a rational drug combination for treating this aggressive human malignancy. PMID:17363738

  6. The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway.

    PubMed

    Sjölin-Goodfellow, Hanna; Frushicheva, Maria P; Ji, Qinqin; Cheng, Debra A; Kadlecek, Theresa A; Cantor, Aaron J; Kuriyan, John; Chakraborty, Arup K; Salomon, Arthur R; Weiss, Arthur

    2015-05-19

    T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain-associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry-based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity. The data indicated that the kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ chain components of the TCR and of signaling molecules downstream of Lck, including ZAP-70. We developed a computational model that provides a mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70-deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporated negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and predicted the order in which tyrosines in the ITAMs of TCR ζ chains must be phosphorylated to be consistent with the experimental data. PMID:25990959

  7. Interleukin 6 Is Required for Pancreatic Cancer Progression by Promoting MAPK Signaling Activation and Oxidative Stress Resistance

    PubMed Central

    Zhang, Yaqing; Yan, Wei; Collins, Meredith A.; Bednar, Filip; Rakshit, Sabita; Zetter, Bruce R.; Stanger, Ben Z.; Chung, Ivy; Rhim, Andrew D.; di Magliano, Marina Pasca

    2013-01-01

    Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with the presence of an oncogenic form of Kras. Mice expressing oncogenic Kras in the pancreas recapitulate the step-wise progression of the human disease. The inflammatory cytokine interleukin 6 (IL6) is often expressed by multiple cell types within the tumor microenvironment. Here, we show that IL6 is required for the maintenance and progression of pancreatic cancer precursor lesions. In fact, the lack of IL6 completely ablates cancer progression even in presence of oncogenic Kras. Mechanistically, we show that IL6 synergizes with oncogenic Kras to activate the reactive oxygen species (ROS) detoxification program downstream of the MAPK/ERK signaling cascade. In addition, IL6 regulates the inflammatory microenvironment of pancreatic cancer throughout its progression, providing several signals that are essential for carcinogenesis. Thus, IL6 emerges as a key player at all stages of pancreatic carcinogenesis, and a potential therapeutic target. PMID:24097820

  8. Mitogen Activated Protein Kinase Family Proteins and c-jun Signaling in Injury-induced Schwann Cell Plasticity.

    PubMed

    Lee, Hye Jeong; Shin, Yoon Kyung; Park, Hwan Tae

    2014-06-01

    Schwann cells (SCs) in the peripheral nerves myelinate axons during postnatal development to allow saltatory conduction of nerve impulses. Well-organized structures of myelin sheathes are maintained throughout life unless nerves are insulted. After peripheral nerve injury, unidentified signals from injured nerves drive SC dedifferentiation into an immature state. Dedifferentiated SCs participate in axonal regeneration by producing neurotrophic factors and removing degenerating nerve debris. In this review, we focus on the role of mitogen activated protein kinase family proteins (MAP kinases) in SC dedifferentiation. In addition, we will highlight neuregulin 1 and the transcription factor c-jun as upstream and downstream signals for MAP kinases in SC responses to nerve injury. PMID:24963277

  9. Chemical Signaling and Functional Activation in Colloidosome-Based Protocells.

    PubMed

    Sun, Shiyong; Li, Mei; Dong, Faqin; Wang, Shengjie; Tian, Liangfei; Mann, Stephen

    2016-04-13

    An aqueous-based microcompartmentalized model involving the integration of partially hydrophobic Fe(III)-rich montmorillonite (FeM) clay particles as structural and catalytic building blocks for colloidosome membrane assembly, self-directed membrane remodeling, and signal-induced protocell communication is described. The clay colloidosomes exhibit size- and charge-selective permeability, and show dual catalytic functions involving spatially confined enzyme-mediated dephosphorylation and peroxidase-like membrane activity. The latter is used for the colloidosome-mediated synthesis and assembly of a temperature-responsive poly(N-isopropylacrylamide)(PNIPAM)/clay-integrated hybrid membrane. In situ PNIPAM elaboration of the membrane is coupled to a glucose oxidase (GOx)-mediated signaling pathway to establish a primitive model of chemical communication and functional activation within a synthetic "protocell community" comprising a mixed population of GOx-containing silica colloidosomes and alkaline phosphatase (ALP)-containing FeM-clay colloidosomes. Triggering the enzyme reaction in the silica colloidosomes gives a hydrogen peroxide signal that induces polymer wall formation in a coexistent population of the FeM-clay colloidosomes, which in turn generates self-regulated membrane-gated ALP-activity within the clay microcompartments. The emergence of new functionalities in inorganic colloidosomes via chemical communication between different protocell populations provides a first step toward the realization of interacting communities of synthetic functional microcompartments. PMID:26923794

  10. Downstream in Mawrth Valles

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site]

    The THEMIS VIS camera is capable of capturing color images of the Martian surface using five different color filters. In this mode of operation, the spatial resolution and coverage of the image must be reduced to accommodate the additional data volume produced from using multiple filters. To make a color image, three of the five filter images (each in grayscale) are selected. Each is contrast enhanced and then converted to a red, green, or blue intensity image. These three images are then combined to produce a full color, single image. Because the THEMIS color filters don't span the full range of colors seen by the human eye, a color THEMIS image does not represent true color. Also, because each single-filter image is contrast enhanced before inclusion in the three-color image, the apparent color variation of the scene is exaggerated. Nevertheless, the color variation that does appear is representative of some change in color, however subtle, in the actual scene. Note that the long edges of THEMIS color images typically contain color artifacts that do not represent surface variation.

    This false color image is from further downstream in Mawrth Valles than yesterday's image. The channel here is at the end of the vallis. This image was collected during the Northern Spring season.

    Image information: VIS instrument. Latitude 26.7, Longitude 340.2 East (19.8 West). 37 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages

  11. Wnt Signaling Activates Shh Signaling in Early Postnatal Intervertebral Discs, and Re-Activates Shh Signaling in Old Discs in the Mouse

    PubMed Central

    Sinner, Debora; Wylie, Christopher C.; Dahia, Chitra Lekha

    2014-01-01

    Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease. PMID:24892825

  12. Exploiting the yeast stress-activated signaling network to inform on stress biology and disease signaling

    PubMed Central

    Ho, Yi-Hsuan

    2016-01-01

    Healthy cells utilize intricate systems to monitor their environment and mount robust responses in the event of cellular stress. Whether stress arises from external insults or defects due to mutation and disease, cells must be able to respond precisely to mount the appropriate defenses. Multi-faceted stress responses are generally coupled with arrest of growth and cell-cycle progression, which both limits the transmission of damaged materials and serves to reallocate limited cellular resources toward defense. Therefore, stress defense versus rapid growth represent competing interests in the cell. How eukaryotic cells set the balance between defense versus proliferation, and in particular knowledge of the regulatory networks that control this decision, are poorly understood. In this perspective, we expand upon our recent work inferring the stress-activated signaling network in budding yeast, which captures pathways controlling stress defense and regulators of growth and cell-cycle progression. We highlight similarities between the yeast and mammalian stress responses and explore how stress-activated signaling networks in yeast can inform on signaling defects in human cancers. PMID:25957506

  13. The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway

    PubMed Central

    Cheng, Debra A; Kadlecek, Theresa A.; Cantor, Aaron J.; Kuriyan, John

    2015-01-01

    T cell activation must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The mechanisms controlling the fine-tuning of T cell receptor (TCR) signaling and T cell activation are unclear. The Syk family kinase ζ chain–associated protein kinase of 70 kD (ZAP-70) is a critical component of the TCR signaling machinery that leads to T cell activation. To elucidate potential feedback targets that are dependent on the kinase activity of ZAP-70, we performed a mass spectrometry–based, phosphoproteomic study to quantify temporal changes in phosphorylation patterns after inhibition of ZAP-70 catalytic activity. Our results provide insights into the fine-tuning of the T cell signaling network before and after TCR engagement. The data indicate that the kinase activity of ZAP-70 stimulates negative feedback pathways that target the Src family kinase Lck and modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chain components of the TCR, and of downstream signaling molecules, including ZAP-70. We developed a computational model that provides a unified mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70–deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporates negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and makes unanticipated specific predictions for the order in which tyrosines in the ITAMs of TCR ζ-chains must be phosphorylated to be consistent with the experimental data. PMID:25990959

  14. RalA, a GTPase targeted by miR-181a, promotes transformation and progression by activating the Ras-related signaling pathway in chronic myelogenous leukemia

    PubMed Central

    Luo, Xiaochuang; Yang, Juhua; Li, Yumin; Li, Tianfu; Wang, Ruirui; Fei, Jia

    2016-01-01

    BCR/ABL is a well-known activator of multiple signaling pathways. RalA, a Ras downstream signaling molecule and a small GTPase, plays an important role in Bcr-Abl-induced leukemogenesis but the exact mechanism remains elusive. Here, we show that RalA GTPase activity is commonly high in chronic myelogenous leukemia (CML) cell lines and patient samples. Overexpression of RalA results in malignant transformation and progression, and induces resistance to imatinib (IM) in BaF3 and K562 cell lines. RalA reduced survival and led to IM resistance in a xenografted mouse model. Ablation of RalA by either siRNA or miR-181a, a RalA targeting microRNA, attenuated the malignant phenotypes in K562 cells. RBC8, a selective Ral inhibitor, enhanced the inhibitory effects of IM in K562, KCL22 and BaF3-P210 cells. Interestingly, the phospho-specific protein microarray assay revealed that multiple phosphorylation signal proteins were decreased by RalA inhibition, including SAPK, JNK, SRC, VEGFR2, P38 MAPK, c-Kit, JunB, and Keratin18. Among them, P38 MAPK and SAPK/JNK are Ras downstream signaling kinases. Taken together, RalA GTPase might be an important oncogene activating the Ras-related signaling pathway in CML. PMID:26967392

  15. Studies on mitogen-activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats.

    PubMed

    Huang, Yan; Meng, Xiao-Ming; Jiang, Guo-Lin; Yang, Ya-Ru; Liu, Juan; Lv, Xiong-Wen; Li, Jun

    2015-02-01

    Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in alveolar macrophages (AMs), activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the present study, we investigated the MAPK pathway in AMs of chronic bronchitis (CB) rats. CB was induced by endotracheal instillation of LPS followed by Bacillus Calmette Guerin injection through the caudal vein 1 week later. Specific inhibitors were used and protein phosphorylations were detected by Western blot. We found that Genistein (PTK inhibitor) could inhibit protein kinase C (PKC), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt or PKB) MAPK signaling pathway with different degrees, LY294002 (PI3K inhibitor) could not only inhibit phospho-PI3K/Akt expression, but also inhibit p38 and c-Jun NH2-terminal kinases (JNK) phosphorylation. Calphostin C (PKC inhibitor) could inhibit phospho-PKC expression and exerted significant effects on extracellular signal-regulated kinases (ERK) phosphorylation, however, it had no impact on p38 and JNK phosphorylation. These results demonstrated that the LPS mediated signaling pathway of MAPK in AMs of CB rats could be described as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3K-PKC-ERK, and PI3K may have a negative regulation on the activation of downstream proteins. PMID:25467375

  16. Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer.

    PubMed

    Hu, Peizhen; Chu, Gina C-Y; Zhu, Guodong; Yang, Hua; Luthringer, Daniel; Prins, Gail; Habib, Fouad; Wang, Yuzhuo; Wang, Ruoxiang; Chung, Leland W K; Zhau, Haiyen E

    2011-01-01

    The potential application of multiplexed quantum dot labeling (MQDL) for cancer detection and prognosis and monitoring therapeutic responses has attracted the interests of bioengineers, pathologists and cancer biologists. Many published studies claim that MQDL is effective for cancer biomarker detection and useful in cancer diagnosis and prognosis, these studies have not been standardized against quantitative biochemical and molecular determinations. In the present study, we used a molecularly characterized human prostate cancer cell model exhibiting activated c-Met signaling with epithelial to mesenchymal transition (EMT) and lethal metastatic progression to bone and soft tissues as the gold standard, and compared the c-Met cell signaling network in this model, in clinical human prostate cancer tissue specimens and in a castration-resistant human prostate cancer xenograft model. We observed c-Met signaling network activation, manifested by increased phosphorylated c-Met in all three. The downstream survival signaling network was mediated by NF-κB and Mcl-1 and EMT was driven by receptor activator of NF-κB ligand (RANKL), at the single cell level in clinical prostate cancer specimens and the xenograft model. Results were confirmed by real-time RT-PCR and western blots in a human prostate cancer cell model. MQDL is a powerful tool for assessing biomarker expression and it offers molecular insights into cancer progression at both the cell and tissue level with high degree of sensitivity. PMID:22205960

  17. Expression of nuclear factor of activated T cells (NFAT) and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during hibernation.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-01-01

    The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through

  18. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc. PMID:27563853

  19. Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module*

    PubMed Central

    Glatz, Gábor; Gógl, Gergő; Alexa, Anita; Reményi, Attila

    2013-01-01

    Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins. PMID:23382384

  20. Limits of downstream hydraulic geometry

    NASA Astrophysics Data System (ADS)

    Wohl, Ellen

    2004-10-01

    Adjustments to flow width, depth, and velocity in response to changes in discharge are commonly characterized by using downstream hydraulic geometry relationships. The spatial limits of these relationships within a drainage basin have not been systematically quantified. Where the erosional resistance of the channel substrate is sufficiently large, hydraulic driving forces presumably will be unable to adjust channel form. Data sets from 10 mountain rivers in the United States, Panama, Nepal, and New Zealand are used in this study to explore the limits of downstream hydraulic geometry relationships. Where the ratio of stream power to sediment size (Ω/D84) exceeds 10,000 kg/s3, downstream hydraulic geometry is well developed; where the ratio falls below 10,000 kg/s3, downstream hydraulic geometry relationships are poorly developed. These limitations on downstream hydraulic geometry have important implications for channel engineering and simulations of landscape change.

  1. mTORC1 signaling activates NRF1 to increase cellular proteasome levels

    PubMed Central

    Zhang, Yinan; Manning, Brendan D

    2015-01-01

    Defects in the maintenance of protein homeostasis, or proteostasis, has emerged as an underlying feature of a variety of human pathologies, including aging-related diseases. Proteostasis is achieved through the coordinated action of cellular systems overseeing amino acid availability, mRNA translation, protein folding, secretion, and degradation. The regulation of these distinct systems must be integrated at various points to attain a proper balance. In a recent study, we found that the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) pathway, well known to enhance the protein synthesis capacity of cells while concordantly inhibiting autophagy, promotes the production of more proteasomes. Activation of mTORC1 genetically, through loss of the tuberous sclerosis complex (TSC) tumor suppressors, or physiologically, through growth factors or feeding, stimulates a transcriptional program involving the sterol-regulatory element binding protein 1 (SREBP1) and nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1) transcription factors leading to an increase in cellular proteasome content. As discussed here, our findings suggest that this increase in proteasome levels facilitates both the maintenance of proteostasis and the recovery of amino acids in the face of an increased protein load consequent to mTORC1 activation. We also consider the physiological and pathological implications of this unexpected new downstream branch of mTORC1 signaling. PMID:26017155

  2. Rice bran feruloylated oligosaccharides activate dendritic cells via Toll-like receptor 2 and 4 signaling.

    PubMed

    Lin, Chi Chen; Chen, Hua Han; Chen, Yu Kuo; Chang, Hung Chia; Lin, Ping Yi; Pan, I-Hong; Chen, Der-Yuan; Chen, Chuan Mu; Lin, Su Yi

    2014-01-01

    This work presents the effects of feruloylated oligosaccharides (FOs) of rice bran on murine bone marrow-derived dendritic cells (BMDCs) and the potential pathway through which the effects are mediated. We found that FOs induced phenotypic maturation of DCs, as shown by the increased expression of CD40, CD80/CD86 and MHC-I/II molecules. FOs efficiently induced maturation of DCs generated from C3H/HeN or C57BL/6 mice with normal toll-like receptor 4 (TLR-4) or TLR-2 but not DCs from mice with mutated TLR4 or TLR2. The mechanism of action of FOs may be mediated by increased phosphorylation of ERK, p38 and JNK mitogen-activated protein kinase (MAPKs) and increased NF-kB activity, which are important signaling molecules downstream of TLR-4 and TLR-2. These data suggest that FOs induce DCs maturation through TLR-4 and/or TLR-2 and that FOs might have potential efficacy against tumor or virus infection or represent a candidate-adjuvant approach for application in immunotherapy and vaccination. PMID:24762969

  3. Differences in Cell Death Induction by Phytophthora Elicitins Are Determined by Signal Components Downstream of MAP Kinase Kinase in Different Species of Nicotiana and Cultivars of Brassica rapa and Raphanus sativus[w

    PubMed Central

    Takemoto, Daigo; Hardham, Adrienne R.; Jones, David A.

    2005-01-01

    Elicitins are small, secreted proteins produced by species of the plant-pathogenic oomycete Phytophthora. They induce hypersensitive cell death in most Nicotiana species and in some cultivars of Brassica rapa and Raphanus sativus. In this study, two true-breeding Fast Cycling B. rapa lines were established that showed severe necrosis (line 7-R) or no visible response (line 18-NR) after treatment with elicitin. Unexpectedly, microscopic examination revealed localized cell death in line 18-NR plants, and expression levels of various defense-marker genes were comparable in both lines. These results suggested that both “responsive” and “nonresponsive” plants responded to elicitin but differed in the extent of the cell death response. Expression of a constitutively active form of Arabidopsis (Arabidopsis thaliana) MAP kinase kinase 4 (AtMEK4DD) also induced rapid development of confluent cell death in line 7-R, whereas line 18-NR showed no visible cell death. Similarly, elicitin-responsive Nicotiana species and R. sativus cultivars showed significantly stronger cell death responses following expression of AtMEK4DD compared with nonresponsive species/cultivars. Line 7-R also showed higher sensitivity to toxin-containing culture filtrates produced by Alternaria brassicicola, and toxin sensitivity cosegregated with elicitin responsiveness, suggesting that the downstream responses induced by elicitin and Alternaria toxin share factors that control the extent of cell death. Interestingly, elicitin responsiveness was shown to correlate with greater susceptibility to A. brassicicola (a necrotroph) in B. rapa but less susceptibility to Phytophthora nicotianae (a hemibiotroph) in Nicotiana, suggesting a more extensive cell death response could cause opposite effects on the outcomes of biotrophic versus necrotrophic plant-pathogen interactions. PMID:15980203

  4. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    SciTech Connect

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  5. Signal integration by Ca2+ regulates intestinal stem cell activity

    PubMed Central

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  6. Robust Indoor Human Activity Recognition Using Wireless Signals

    PubMed Central

    Wang, Yi; Jiang, Xinli; Cao, Rongyu; Wang, Xiyang

    2015-01-01

    Wireless signals–based activity detection and recognition technology may be complementary to the existing vision-based methods, especially under the circumstance of occlusions, viewpoint change, complex background, lighting condition change, and so on. This paper explores the properties of the channel state information (CSI) of Wi-Fi signals, and presents a robust indoor daily human activity recognition framework with only one pair of transmission points (TP) and access points (AP). First of all, some indoor human actions are selected as primitive actions forming a training set. Then, an online filtering method is designed to make actions’ CSI curves smooth and allow them to contain enough pattern information. Each primitive action pattern can be segmented from the outliers of its multi-input multi-output (MIMO) signals by a proposed segmentation method. Lastly, in online activities recognition, by selecting proper features and Support Vector Machine (SVM) based multi-classification, activities constituted by primitive actions can be recognized insensitive to the locations, orientations, and speeds. PMID:26184231

  7. Dichloromethane fraction of Cimicifuga heracleifolia decreases the level of melanin synthesis by activating the ERK or AKT signaling pathway in B16F10 cells.

    PubMed

    Jang, Ji Yeon; Lee, Jun Hyuk; Kang, Byoung Won; Chung, Kyung Tae; Choi, Yung Hyun; Choi, Byung Tae

    2009-03-01

    Cimicifuga rhizoma has long been used in traditional Korean medicine. In particular, a Cimicifuga heracleifolia extract (CHE) was reported to inhibit the formation of glutamate and the glutamate dehydrogenase activity in cultured rat islet. Glutamate activates melanogenesis by activating tyrosinase. Accordingly, it was hypothesized that a CHE might inhibit the melanogenesis-related signal pathways including the inhibition of microphthalmia-associated transcription factor (MITF)-tyrosinase signaling and/or the activation of extracellular signal-regulated kinase (ERK)-Akt signaling. The results showed that CHE inhibits the cellular melanin contents, tyrosinase activity and expression of melanogenesis-related proteins including MITF, tyrosinase and tyrosinase-related protein (TRP)s in alpha-melanocyte-stimulating hormone-stimulated B16 cells. Moreover, CHE phosphorylates MEK, ERK1/2 and Akt, which are melanogenesis inhibitory proteins. The data suggest that CHE inhibits melanogenesis signaling by both inhibiting the tyrosinase directly and activating the MEK-ERK or Akt signal pathways-mediated suppression of MITF and its downstream signal pathway, including tyrosinase and TRPs. Therefore, C. heracleifolia would be a useful therapeutic agent for treating hyperpigmentation and an effective component in whitening and/or lightening cosmetics. PMID:18803655

  8. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    PubMed Central

    Lina, Taslima T.; Dunphy, Paige S.; Luo, Tian

    2016-01-01

    ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. PMID:27381289

  9. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  10. Cutting edge: An in vivo reporter reveals active B cell receptor signaling in the germinal center.

    PubMed

    Mueller, James; Matloubian, Mehrdad; Zikherman, Julie

    2015-04-01

    Long-lasting Ab responses rely on the germinal center (GC), where B cells bearing high-affinity Ag receptors are selected from a randomly mutated pool to populate the memory and plasma cell compartments. Signaling downstream of the BCR is dampened in GC B cells, raising the possibility that Ag presentation and competition for T cell help, rather than Ag-dependent signaling per se, drive these critical selection events. In this study we use an in vivo reporter of BCR signaling, Nur77-eGFP, to demonstrate that although BCR signaling is reduced among GC B cells, a small population of cells exhibiting GC light zone phenotype (site of Ag and follicular helper T cell encounter) express much higher levels of GFP. We show that these cells exhibit somatic hypermutation, gene expression characteristic of signaling and selection, and undergo BCR signaling in vivo. PMID:25725108

  11. Pathway connectivity and signaling coordination in the yeast stress-activated signaling network

    PubMed Central

    Chasman, Deborah; Ho, Yi-Hsuan; Berry, David B; Nemec, Corey M; MacGilvray, Matthew E; Hose, James; Merrill, Anna E; Lee, M Violet; Will, Jessica L; Coon, Joshua J; Ansari, Aseem Z; Craven, Mark; Gasch, Audrey P

    2014-01-01

    Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology. PMID:25411400

  12. Angiotensin II activates different calcium signaling pathways in adipocytes.

    PubMed

    Dolgacheva, Lyudmila P; Turovskaya, Maria V; Dynnik, Vladimir V; Zinchenko, Valery P; Goncharov, Nikolay V; Davletov, Bazbek; Turovsky, Egor A

    2016-03-01

    Angiotensin II (Ang II) is an important mammalian neurohormone involved in reninangiotensin system. Ang II is produced both constitutively and locally by RAS systems, including white fat adipocytes. The influence of Ang II on adipocytes is complex, affecting different systems of signal transduction from early Са(2+) responses to cell proliferation and differentiation, triglyceride accumulation, expression of adipokine-encoding genes and adipokine secretion. It is known that white fat adipocytes express all RAS components and Ang II receptors (АТ1 and АТ2). The current work was carried out with the primary white adipocytes culture, and Са(2+) signaling pathways activated by Ang II were investigated using fluorescent microscopy. Са(2+)-oscillations and transient responses of differentiated adipocytes to Ang II were registered in cells with both small and multiple lipid inclusions. Using inhibitory analysis and selective antagonists, we now show that Ang II initiates periodic Са(2+)-oscillations and transient responses by activating АТ1 and АТ2 receptors and involving branched signaling cascades: 1) Ang II → Gq → PLC → IP3 → IP3Rs → Ca(2+) 2) Gβγ → PI3Kγ → PKB 3) PKB → eNOS → NO → PKG 4) CD38 → cADPR → RyRs → Ca(2+) In these cascades, AT1 receptors play the leading role. The results of the present work open a perspective of using Ang II for correction of signal resistance of adipocytes often observed during obesity and type 2 diabetes. PMID:26850364

  13. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  14. Endothelin signaling activates Mef2c expression in the neural crest through a MEF2C-dependent positive-feedback transcriptional pathway.

    PubMed

    Hu, Jianxin; Verzi, Michael P; Robinson, Ashley S; Tang, Paul Ling-Fung; Hua, Lisa L; Xu, Shan-Mei; Kwok, Pui-Yan; Black, Brian L

    2015-08-15

    Endothelin signaling is essential for neural crest development, and dysregulated Endothelin signaling is associated with several neural crest-related disorders, including Waardenburg and other syndromes. However, despite the crucial roles of this pathway in neural crest development and disease, the transcriptional effectors directly activated by Endothelin signaling during neural crest development remain incompletely elucidated. Here, we establish that the MADS box transcription factor MEF2C is an immediate downstream transcriptional target and effector of Endothelin signaling in the neural crest. We show that Endothelin signaling activates Mef2c expression in the neural crest through a conserved enhancer in the Mef2c locus and that CRISPR-mediated deletion of this Mef2c neural crest enhancer from the mouse genome abolishes Endothelin induction of Mef2c expression. Moreover, we demonstrate that Endothelin signaling activates neural crest expression of Mef2c by de-repressing MEF2C activity through a Calmodulin-CamKII-histone deacetylase signaling cascade. Thus, these findings identify a MEF2C-dependent, positive-feedback mechanism for Endothelin induction and establish MEF2C as an immediate transcriptional effector and target of Endothelin signaling in the neural crest. PMID:26160899

  15. Over-expression of CKS1B activates both MEK/ERK and JAK/STAT3 signaling pathways and promotes myeloma cell drug-resistance

    PubMed Central

    Zangari, Maurizio; Xu, Hongwei; Cao, Thai M.; Xu, Chunjiao; Wu, Yong; Xiao, Fang; Liu, Yinghong; Yang, Ye; Salama, Mohamed; Li, Guiyuan; Tricot, Guido; Zhan, Fenghuang

    2010-01-01

    Here we demonstrate the crucial role of CKS1B in multiple myeloma (MM) progression and define CKS1B-mediated SKP2/p27Kip1-independent down-stream signaling pathways. Forced-expression of CKS1B in MM cells increased cell multidrug-resistance. CKS1B activates STAT3 and MEK/ERK pathways. In contrast, SKP2 knockdown or p27Kip1 over-expression resulted in activation of the STAT3 and MEK/ERK pathways. Further investigations showed that BCL2 is a downstream target of MEK/ERK signaling. Stimulation of STAT3 and MEK/ERK signaling pathways partially abrogated CKS1B knockdown induced MM cell death and growth inhibition. Targeting STAT3 and MEK/ ERK signaling pathways by specific inhibitors induced significant MM cell death and growth inhibition in CKS1B-overexpressing MM cells and their combinations resulted in synergy. Thus, our findings provide a rationale for targeting STAT3 and MEK/ERK/ BCL2 signaling in aggressive CKS1B-overexpressing MM. PMID:20930946

  16. Digital signaling decouples activation probability and population heterogeneity

    PubMed Central

    Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz; Quake, Stephen R; Tay, Savaş

    2015-01-01

    Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.08931.001 PMID:26488364

  17. Digital signaling decouples activation probability and population heterogeneity.

    PubMed

    Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz; Quake, Stephen R; Tay, Savaş

    2015-01-01

    Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity. PMID:26488364

  18. Activity-Based Probe for Histidine Kinase Signaling

    PubMed Central

    Wilke, Kaelyn E.; Francis, Samson; Carlson, Erin E.

    2012-01-01

    Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins, a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on a RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria including development, virulence and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analog, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, ATPγS-BODIPY, labels active HK proteins and is competitive for the ATP-binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification. PMID:22606938

  19. Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels

    PubMed Central

    Qi, Zhi; Verma, Rajeev; Gehring, Chris; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

    2010-01-01

    A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca2+ permeable channels in mesophyll cells, resulting in cytosolic Ca2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca2+ conductance and resulting cytosolic Ca2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca2+-dependent manner. PMID:21088220

  20. RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity

    PubMed Central

    Kawasaki, Jun; Aegerter, Sandrine; Fevurly, R. Dawn; Mammoto, Akiko; Mammoto, Tadanori; Sahin, Mustafa; Mably, John D.; Fishman, Steven J.; Chan, Joanne

    2014-01-01

    Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations. PMID:24837431

  1. Digital Signal Processing System for Active Noise Reduction

    NASA Astrophysics Data System (ADS)

    Edmonson, William W.; Tucker, Jerry

    2002-12-01

    Over the years there has been a need to improve the comfort of passengers in flight. One avenue for increasing comfort is to reduce cabin noise that is attributed to the engine and the vibration of fuselage panels that radiate sound. High frequency noise can be abated using sound absorbing material. Though, for low frequency noise the sound absorption material would have to very thick, thereby reducing the cabin size. To reduce these low frequency disturbances, active noise control systems (ANC) is being developed that utilizes feedback for cancellation of the disturbance. The active noise control system must be small in size, be a low power device, and operate in real-time. It must also be numerically stable i.e. insensitive to temperature and pressure variations. The ANC system will be a module that consists of digital signal processor (DSP), analog-digital and digital-analog converters, power converters, an actuator and sensors. The DSP will implement the feedback control algorithm that controls the actuators. This module will be attached to panels on the inside of the fuselage for actively eliminating resonant modes of the structure caused by turbulent flow across the fuselage Skin. A hardware prototype of the ANC system must be able to eliminate broadband noise consisting of a bandwidth between 100 Hz and 1500 Hz, which requires a sample rate of 5000 Hz. The analog/digital converters output accuracy is 16 bits with a 2's-compliment format and a very short acquisition time. This will also yield the appropriate dynamic range. Similar specifications are required of the digital/analog converter. The processor section of the system integrates a digital signal processor (TI TMS320C33) with analog/digital (Burr-Brown ADS8320) and digital/analog signal (DAC853 1) converters. The converters with associated power conditioning circuitry and test points reside on a daughter board that sits on top of a Spectrum Digital evaluation module. This will have the ability to test

  2. The role of the Janus kinase family/signal transducer and activator of transcription signaling pathway in fibrotic renal disease

    PubMed Central

    Matsui, Futoshi; Meldrum, Kirstan K.

    2012-01-01

    Over the past several years, a number of cytokines and growth factors including transforming growth factor β1, tumor necrosis factor α, and angiotensin II have been shown to play a crucial role in renal fibrosis. The Janus kinase family (JAK) and signal transducers and activators of transcription (STATs) constitute one of the primary signaling pathways that regulate cytokine expression, and the JAK/STAT signaling pathway has increasingly been implicated in the pathophysiology of renal disease. This review examines the role of the JAK/STAT signaling pathway in fibrotic renal disease. The JAK/STAT signaling pathway is activated in a variety of renal diseases and has been implicated in the pathophysiology of renal fibrosis. Experimental evidence suggests that inhibition of the JAK/STAT signaling pathway, in particular JAK2 and STAT3, may suppress renal fibrosis and protect renal function. However, it is incompletely understood which cells activate the JAK/STAT signaling pathway and which JAK/STAT signaling pathway is activated in each renal disease. Research regarding JAK/STAT signaling and its contribution to renal disease is still ongoing in humans. Future studies are required to elucidate the potential role of JAK/STAT signaling inhibition as a therapeutic strategy in the attenuation of renal fibrosis. PMID:22883438

  3. Role of Calcium Signaling in B Cell Activation and Biology.

    PubMed

    Baba, Yoshihiro; Kurosaki, Tomohiro

    2016-01-01

    Increase in intracellular levels of calcium ions (Ca2+) is one of the key triggering signals for the development of B cell response to the antigen. The diverse Ca2+ signals finely controlled by multiple factors participate in the regulation of gene expression, B cell development, and effector functions. B cell receptor (BCR)-initiated Ca2+ mobilization is sourced from two pathways: one is the release of Ca2+ from the intracellular stores, endoplasmic reticulum (ER), and other is the prolonged influx of extracellular Ca2+ induced by depleting the stores via store-operated calcium entry (SOCE) and calcium release-activated calcium (CRAC) channels. The identification of stromal interaction molecule 1(STIM1), the ER Ca2+ sensor, and Orai1, a key subunit of the CRAC channel pore, has now provided the tools to understand the mode of Ca2+ influx regulation and physiological relevance. Herein, we discuss our current understanding of the molecular mechanisms underlying BCR-triggered Ca2+ signaling as well as its contribution to the B cell biological processes and diseases. PMID:26369772

  4. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  5. The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

    PubMed Central

    Baggio, L; Morrison, M

    1996-01-01

    Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. PMID:8955404

  6. Transcriptional regulatory elements downstream of the JunB gene.

    PubMed Central

    Perez-Albuerne, E D; Schatteman, G; Sanders, L K; Nathans, D

    1993-01-01

    JunB is an immediate early transcription factor that is induced by a variety of extracellular signaling agents, including growth factors, phorbol esters, and agents that elevate cyclic AMP. The mechanism of activation of the gene encoding JunB by these agents is not well understood. By using the JunB gene together with flanking DNA in transfection experiments, we show that a serum response element (SRE) and/or a cAMP response element (CRE) downstream of the gene mediate the response of the gene in mouse NIH 3T3 cells to serum, platelet-derived growth factor, basic fibroblast growth factor, phorbol ester, and forskolin. In addition, a segment of DNA just upstream of the TATA box is required for optimal activation of the gene. Images Fig. 1 Fig. 5 PMID:8265655

  7. Transsynaptic signaling by activity-dependent cleavage of neuroligin-1.

    PubMed

    Peixoto, Rui T; Kunz, Portia A; Kwon, Hyungbae; Mabb, Angela M; Sabatini, Bernardo L; Philpot, Benjamin D; Ehlers, Michael D

    2012-10-18

    Adhesive contact between pre- and postsynaptic neurons initiates synapse formation during brain development and provides a natural means of transsynaptic signaling. Numerous adhesion molecules and their role during synapse development have been described in detail. However, once established, the mechanisms of adhesive disassembly and its function in regulating synaptic transmission have been unclear. Here, we report that synaptic activity induces acute proteolytic cleavage of neuroligin-1 (NLG1), a postsynaptic adhesion molecule at glutamatergic synapses. NLG1 cleavage is triggered by NMDA receptor activation, requires Ca2+ /calmodulin-dependent protein kinase, and is mediated by proteolytic activity of matrix metalloprotease 9 (MMP9). Cleavage of NLG1 occurs at single activated spines, is regulated by neural activity in vivo, and causes rapid destabilization of its presynaptic partner neurexin-1β (NRX1β). In turn, NLG1 cleavage depresses synaptic transmission by abruptly reducing presynaptic release probability. Thus, local proteolytic control of synaptic adhesion tunes synaptic transmission during brain development and plasticity. PMID:23083741

  8. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

    PubMed Central

    Medina-Castellanos, Elizabeth; Esquivel-Naranjo, Edgardo U.; Heil, Martin; Herrera-Estrella, Alfredo

    2014-01-01

    The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals. PMID:25484887

  9. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell.

    PubMed

    Xue, Shaowu; Hu, Honghong; Ries, Amber; Merilo, Ebe; Kollist, Hannes; Schroeder, Julian I

    2011-04-20

    Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented. PMID:21423149

  10. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    SciTech Connect

    Ohashi, Kazuya; Nagata, Yosuke; Wada, Eiji; Zammit, Peter S.; Shiozuka, Masataka; Matsuda, Ryoichi

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  11. Stromal Hedgehog signalling is downregulated in colon cancer and its restoration restrains tumour growth.

    PubMed

    Gerling, Marco; Büller, Nikè V J A; Kirn, Leonard M; Joost, Simon; Frings, Oliver; Englert, Benjamin; Bergström, Åsa; Kuiper, Raoul V; Blaas, Leander; Wielenga, Mattheus C B; Almer, Sven; Kühl, Anja A; Fredlund, Erik; van den Brink, Gijs R; Toftgård, Rune

    2016-01-01

    A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor. PMID:27492255

  12. Stromal Hedgehog signalling is downregulated in colon cancer and its restoration restrains tumour growth

    PubMed Central

    Gerling, Marco; Büller, Nikè V. J. A.; Kirn, Leonard M.; Joost, Simon; Frings, Oliver; Englert, Benjamin; Bergström, Åsa; Kuiper, Raoul V.; Blaas, Leander; Wielenga, Mattheus C. B.; Almer, Sven; Kühl, Anja A.; Fredlund, Erik; van den Brink, Gijs R.; Toftgård, Rune

    2016-01-01

    A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor. PMID:27492255

  13. A novel nuclear export signal in Smad1 is essential for its signaling activity.

    PubMed

    Xiao, Zhan; Brownawell, Amy M; Macara, Ian G; Lodish, Harvey F

    2003-09-01

    To investigate the subcellular distributions of Smad proteins, the intracellular mediators of transforming growth factor-beta family cytokines, we examined their sequences for nuclear export signals (NES). We found a leucine-rich NES-like motif (termed NES2) in the central linker region of the receptor-regulated Smads that is absent from the other two classes of Smads (Co-Smads and I-Smads). In microinjection assays, NES2 peptide caused nuclear export of a fused glutathione S-transferase protein. Mutations in NES2 converted Smad1 from an even distribution throughout the cells into an exclusive nuclear localization in both transiently and stably expressing cell lines, and this nuclear enrichment was more pronounced than that induced by mutations in NES1. Furthermore, overexpression of CRM1, the cellular export receptor, transforms Smad1 into a mostly cytoplasmic profile by enhancing its nuclear export. The Smad1 NES2 mutant but not the Smad1 NES1 mutant is mostly resistant to this cytoplasmic targeting, indicating that NES2, not NES1, is the major target for CRM1 in Smad1. We further confirmed the functionality of NES2 by a heterokaryon assay. The Smad1 NES1 mutant displays good ligand responsiveness and moderately lowered transcriptional activity compared with wild type Smad1. In contrast, the Smad1 NES2 mutant shows a severe disruption in reporter gene activation, minimal response to bone morphogenetic protein stimulation, and significantly lowered bone morphogenetic protein-induced phosphorylation, which may be the reason for its deficient transcription activity. Thus, we have defined a major NES in Smad1 that is essential for its ligand-induced coupling with cell surface receptors and hence, transcriptional activity. Our study, along with recent studies of the nucleocytoplasmic shuttling of Smad2 and Smad3 proteins, demonstrate that continued nucleocytoplasmic shuttling is a common requisite for the active signaling of R-Smads. Although conserved in other R

  14. Critical Role for an Acidic Amino Acid Region in Platelet Signaling by the HemITAM (Hemi-immunoreceptor Tyrosine-based Activation Motif) Containing Receptor CLEC-2 (C-type Lectin Receptor-2)*

    PubMed Central

    Hughes, Craig E.; Sinha, Uma; Pandey, Anjali; Eble, Johannes A.; O'Callaghan, Christopher A.; Watson, Steve P.

    2013-01-01

    CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors. PMID:23264619

  15. Signal Transducer and Activator of Transcription-3, Inflammation, and Cancer

    PubMed Central

    Aggarwal, Bharat B.; Kunnumakkara, Ajaikumar B.; Harikumar, Kuzhuvelil B.; Gupta, Shan R.; Tharakan, Sheeja T.; Koca, Cemile; Dey, Sanjit; Sung, Bokyung

    2011-01-01

    Signal transducer and activator of transcription-3 (STAT-3) is one of six members of a family of transcription factors. It was discovered almost 15 years ago as an acute-phase response factor. This factor has now been associated with inflammation, cellular transformation, survival, proliferation, invasion, angiogenesis, and metastasis of cancer. Various types of carcinogens, radiation, viruses, growth factors, oncogenes, and inflammatory cytokines have been found to activate STAT-3. STAT-3 is constitutively active in most tumor cells but not in normal cells. Phosphorylation of STAT-3 at tyrosine 705 leads to its dimerization, nuclear translocation, DNA binding, and gene transcription. The phosphorylation of STAT-3 at serine 727 may regulate its activity negatively or positively. STAT-3 regulates the expression of genes that mediate survival (survivin, bcl-xl, mcl-1, cellular FLICE-like inhibitory protein), proliferation (c-fos, c-myc, cyclin D1), invasion (matrix metalloproteinase-2), and angiogenesis (vascular endothelial growth factor). STAT-3 activation has also been associated with both chemoresistance and radioresistance. STAT-3 mediates these effects through its collaboration with various other transcription factors, including nuclear factor-κB, hypoxia-inducible factor-1, and peroxisome proliferator activated receptor-γ. Because of its critical role in tumorigenesis, inhibitors of this factor’s activation are being sought for both prevention and therapy of cancer. This has led to identification of small peptides, oligonucleotides, and small molecules as potential STAT-3 inhibitors. Several of these small molecules are chemo-preventive agents derived from plants. This review discusses the intimate relationship between STAT-3, inflammation, and cancer in more detail. PMID:19723038

  16. Dynamic neural activity during stress signals resilient coping.

    PubMed

    Sinha, Rajita; Lacadie, Cheryl M; Constable, R Todd; Seo, Dongju

    2016-08-01

    Active coping underlies a healthy stress response, but neural processes supporting such resilient coping are not well-known. Using a brief, sustained exposure paradigm contrasting highly stressful, threatening, and violent stimuli versus nonaversive neutral visual stimuli in a functional magnetic resonance imaging (fMRI) study, we show significant subjective, physiologic, and endocrine increases and temporally related dynamically distinct patterns of neural activation in brain circuits underlying the stress response. First, stress-specific sustained increases in the amygdala, striatum, hypothalamus, midbrain, right insula, and right dorsolateral prefrontal cortex (DLPFC) regions supported the stress processing and reactivity circuit. Second, dynamic neural activation during stress versus neutral runs, showing early increases followed by later reduced activation in the ventrolateral prefrontal cortex (VLPFC), dorsal anterior cingulate cortex (dACC), left DLPFC, hippocampus, and left insula, suggested a stress adaptation response network. Finally, dynamic stress-specific mobilization of the ventromedial prefrontal cortex (VmPFC), marked by initial hypoactivity followed by increased VmPFC activation, pointed to the VmPFC as a key locus of the emotional and behavioral control network. Consistent with this finding, greater neural flexibility signals in the VmPFC during stress correlated with active coping ratings whereas lower dynamic activity in the VmPFC also predicted a higher level of maladaptive coping behaviors in real life, including binge alcohol intake, emotional eating, and frequency of arguments and fights. These findings demonstrate acute functional neuroplasticity during stress, with distinct and separable brain networks that underlie critical components of the stress response, and a specific role for VmPFC neuroflexibility in stress-resilient coping. PMID:27432990

  17. Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells

    PubMed Central

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly

    2011-01-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  18. Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells.

    PubMed

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N; Hoek, Jan B; Kiyatkin, Anatoly

    2011-11-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  19. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  20. Aurora A kinase activity influences calcium signaling in kidney cells.

    PubMed

    Plotnikova, Olga V; Pugacheva, Elena N; Golemis, Erica A

    2011-06-13

    Most studies of Aurora A (AurA) describe it as a mitotic centrosomal kinase. However, we and others have recently identified AurA functions as diverse as control of ciliary resorption, cell differentiation, and cell polarity control in interphase cells. In these activities, AurA is transiently activated by noncanonical signals, including Ca(2+)-dependent calmodulin binding. These and other observations suggested that AurA might be involved in pathological conditions, such as polycystic kidney disease (PKD). In this paper, we show that AurA is abundant in normal kidney tissue but is also abnormally expressed and activated in cells lining PKD-associated renal cysts. PKD arises from mutations in the PKD1 or PKD2 genes, encoding polycystins 1 and 2 (PC1 and PC2). AurA binds, phosphorylates, and reduces the activity of PC2, a Ca(2+)-permeable nonselective cation channel and, thus, limits the amplitude of Ca(2+) release from the endoplasmic reticulum. These and other findings suggest AurA may be a relevant new biomarker or target in the therapy of PKD. PMID:21670214

  1. Signal-to-noise ratio in neuro activation PET studies

    SciTech Connect

    Votaw, J.R.

    1996-04-01

    It has become commonplace to compare scanner sensitivity characteristics by comparing noise equivalent count rate curves. However, because a 20-cm diameter uniform phantom is drastically difference from a human brain, these curves give misleading information when planning a neuro activation PET experiment. Signal-to-noise ratio (SNR) calculations have been performed using measured data (Siemens 921 scanner) from the three-dimensional (3-D) Hoffman brain phantom for the purpose of determining the optimal injection and scanning protocol for [{sup 15}O] labeled activation experiments. Region of interest (ROI) values along with the variance due to prompt (trues plus randoms) and random events were determined for various regions and radioactivity concentrations. Calculated attenuation correction was used throughout. Scatter correction was not used when calculating the SNR in activation studies because the number of scattered events is almost identical in each data acquisition and hence cancels. The results indicate that randoms correction should not be performed and that rather than being limited by the scanner capabilities, neuro activation experiments are limited by the amount of radioactivity that can be injected and the length of time the patient can stay in the scanner.

  2. Hypoxic stress: impact on the modulation of TLR2, TLR4, NOD1 and NOD2 receptor and their down-stream signalling genes expression in catla (Catla catla).

    PubMed

    Basu, Madhubanti; Paichha, Mahismita; Lenka, Saswati S; Chakrabarty, Rina; Samanta, Mrinal

    2016-01-01

    The damage-associated molecular patterns (DAMPs) released from the damaged tissue/cells are recently reported as endogenous ligands to activate toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors signaling pathways. In the aquatic environment, reduction in dissolved oxygen (DO) concentration causes hypoxic stress resulting in tissue damage and patho-biological changes in fish. We envisaged the critical role of TLR and NOD receptors in recognizing DAMPs as endogenous ligands during hypoxic stress in fish. Catla (Catla catla) fingerlings (avg. wt ~56 g) was exposed to hypoxic stress (DO: 1-3 mg/L) for 1 and 24 h. After the designated time course, total RNA was extracted from gill, liver, kidney and blood, and modulation of TLRs (TLR2 and TLR4), NOD (NOD1 and NOD2) receptors, MyD88 (myeloid differentiation primary response gene 88), RICK (receptor interacting serine-threonine protein kinase-2), interleukin (IL)-6, IL-8 and IL-10 gene expression were analyzed by quantitative reverse transcriptase PCR assay. Significant (p < 0.05) up-regulation of some DAMPs {high-mobility group box 1 and heat shock protein-70}, TLRs and NOD receptors genes expressions were observed in the hypoxic fish tissues as compared to the control. Further investigation revealed inductive expression of MyD88, RICK, IL-6, IL-8 and IL-10 genes in the TLRs and NODs activated tissues of the hypoxic fish. These data together may suggest the important role of TLRs and NOD receptors signaling pathway in sterile inflammation and pathobiology of fish in hypoxic stress, and warrant further study to investigate the role of TLR and NOD receptors in abiotic stress management in aquaculture. PMID:26590162

  3. The ins and outs of adrenergic signaling.

    PubMed

    Lohse, Martin J

    2015-09-01

    Adrenergic signaling, in particular signaling in the sympathetic nervous system, is a prime example of the control of an essential physiological system. It has served as a model system both for the control of mediator release and for receptor signaling and regulation. This review covers the historical development of the field and then addresses issues that represent key fields of ongoing research: the mechanisms and kinetics of receptor activation, temporal patterns of downstream signaling and signal bias, receptor mobility and aggregation, and signal compartmentation and specificity. The available evidence suggests that adrenergic signaling may involve complex spatiotemporal patterns, which give texture to the signaling process and may contain additional biological information. PMID:26199112

  4. Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells.

    PubMed

    Hasebe, Masaharu; Yoshino, Masami

    2016-06-01

    The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na(+)-activated K(+) (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

  5. URG11 promotes gastric cancer growth and invasion by activation of β-catenin signalling pathway

    PubMed Central

    Du, Rui; Xia, Lin; Sun, Shiren; Lian, Zhaorui; Zou, Xue; Gao, Juan; Xie, Huahong; Fan, Rui; Song, Jiugang; Li, Xiaohua; Liu, Jie; Fan, Daiming

    2010-01-01

    Abstract Upregulated gene 11 (URG11), a new gene upregulated by Heptatitis B Virus X protein (HBx), was previously shown to activate β-catenin and promote hepatocellular growth and tumourigenesis. Although the oncogenic role of URG11 in the development of hepatocellular carcinoma has been well documented, its relevance to other human malignancies and the underlying molecular mechanisms remain largely unknown. Here we reported a novel function of URG11 to promote gastric cancer growth and metastasis. URG11 was found to be highly expressed in gastric cancer tissues compared with adjacent nontumourous ones by immunohistochemical staining and western blot. Knockdown of URG11 expression by small interfering RNA (siRNA) effectively attenuated the proliferation, anchorage-independent growth, invasiveness and metastatic potential of gastric cancer cells. URG11 inhibition led to decreased expression of β-catenin and its nuclear accumulation in gastric cancer cells and extensive costaining between URG11 and β-catenin was observed in gastric cancer tissues. Transient transfection assays with the β-catenin promoter showed that it was inhibited by URG11-specific small inhibitory RNA. Moreover, suppression of endogenous URG11 expression results in decreased activation of β-catenin/TCF and its downstream effector genes, cyclinD1 and membrane type 1 matrix metallopeptidase (MT1-MMP), which are known to be involved in cell proliferation and invasion, respectively. Taken together, our data suggest that URG11 contributes to gastric cancer growth and metastasis at least partially through activation of β-catenin signalling pathway. These findings also propose a promising target for gene therapy in gastric cancer. PMID:19413886

  6. Long-range signaling in growing neurons after local elevation of cyclic AMP-dependent activity

    PubMed Central

    1994-01-01

    Cyclic AMP-dependent activity at the growth cone or the soma of cultured Xenopus spinal neurons was elevated by local extracellular perfusion of the neuron with culture medium containing 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) or forskolin. During local perfusion of one of the growth cones of multipolar neurons with these drugs, the perfused growth cone showed further extension, while the distant, unperfused growth cones were inhibited in their growth. Local perfusion of the growth cone with culture medium or local perfusion with 8-br-cAMP at a cell-free region 100 microns away from the growth cone did not produce any effect on the extension of the growth cone. Reduced extension of all growth cones was observed when the perfusion with 8-br-cAMP was restricted to the soma. The distant inhibitory effect does not depend on the growth of the perfused growth cone since local coperfusion of the growth cone with 8-br-cAMP and colchicine inhibited growth on both perfused and unperfused growth cones, while local perfusion with colchicine alone inhibited only the perfused growth cone. The distant inhibitory effect was abolished when the perfusion of 8-br-cAMP was carried out together with kinase inhibitor H- 8, suggesting the involvement of cAMP-dependent protein kinase and/or its downstream factors in the long-range inhibitory signaling. Uniform exposure of the entire neuron to bath-applied 8-br-cAMP, however, led to enhanced growth activity at all growth cones. Thus, local elevation of cAMP-dependent activity produces long-range and opposite effects on distant parts of the neuron, and a cytosolic gradient of second messengers may produce effects distinctly different from those following uniform global elevation of the messenger, leading to differential growth regulation at different regions of the same neuron. PMID:7798321

  7. Bisphenol A (BPA) stimulates the interferon signaling and activates the inflammasome activity in myeloid cells.

    PubMed

    Panchanathan, Ravichandran; Liu, Hongzhu; Leung, Yuet-Kin; Ho, Shuk-mei; Choubey, Divaker

    2015-11-01

    Environmental factors contribute to the development of autoimmune diseases, including systemic lupus erythematosus (SLE), which exhibits a strong female bias (female-to-male ratio 9:1). However, the molecular mechanisms remain largely unknown. Because a feedforward loop between the female sex hormone estrogen (E2) and type I interferon (IFN-α/β)-signaling induces the expression of certain p200-family proteins (such as murine p202 and human IFI16) that regulate innate immune responses and modify lupus susceptibility, we investigated whether treatment of myeloid cells with bisphenol A (BPA), an environmental estrogen, could regulate the p200-family proteins and activate innate immune responses. We found that treatment of murine bone marrow-derived cells (BMCs) and human peripheral blood mononuclear cells with BPA induced the expression of ERα and IFN-β, activated the IFN-signaling, and stimulated the expression of the p202 and IFI16 proteins. Further, the treatment increased levels of the NLRP3 inflammasome and stimulated its activity. Accordingly, BPA-treatment of BMCs from non lupus-prone C57BL/6 and the lupus-prone (NZB×NZW)F1 mice activated the type I IFN-signaling, induced the expression of p202, and activated an inflammasome activity. Our study demonstrates that BPA-induced signaling in the murine and human myeloid cells stimulates the type I IFN-signaling that results in an induction of the p202 and IFI16 innate immune sensors for the cytosolic DNA and activates an inflammasome activity. These observations provide novel molecular insights into the role of environmental BPA exposures in potentiating the development of certain autoimmune diseases such as SLE. PMID:26277401

  8. Lipoicmethylenedioxyphenol Reduces Experimental Atherosclerosis through Activation of Nrf2 Signaling

    PubMed Central

    Ying, Zhekang; Chen, Minjie; Xie, Xiaoyun; Wang, Xiaoke; Kherada, Nisharahmed; Desikan, Rajagopal; Mihai, Georgeta; Burns, Patrick; Sun, Qinghua; Rajagopalan, Sanjay

    2016-01-01

    Objective Oxidative stress is implicated in the pathogenesis of atherosclerosis, and Nrf2 is the transcriptional factor central in cellular antioxidant responses. In the present study, we investigate the effect of a dihydrolipoic acid derivative lipoicmethylenedioxyphenol (LMDP) on the progression of atherosclerosis and test whether its effect on atherosclerosis is mediated by Nrf2. Methods and Results Both magnetic resonance imaging (MRI) scanning and en face analysis reveal that 14 weeks of treatment with LMDP markedly reduced atherosclerotic burden in a rabbit balloon vascular injury model. Myograph analyses show decreased aortic contractile response to phenylephrine and increased aortic response to acetylcholine and insulin in LMDP-treated animals, suggesting that LMDP inhibits atherosclerosis through improving vascular function. A role of Nrf2 signaling in mediating the amelioration of vascular function by LMDP was supported by increased Nrf2 translocation into nuclear and increased expression of Nrf2 target genes. Furthermore, chemotaxis analysis with Boydem chamber shows that leukocytes isolated from LMDP-treated rabbits had reduced chemotaxis, and knock-down of Nrf2 significantly reduced the effect of LMDP on the chemotaxis of mouse macrophages. Conclusion Our results support that LMDP has an anti-atherosclerotic effect likely through activation of Nrf2 signaling and subsequent inhibition of macrophage chemotaxis. PMID:26859892

  9. Nucleoporin 62-Like Protein Activates Canonical Wnt Signaling through Facilitating the Nuclear Import of β-Catenin in Zebrafish

    PubMed Central

    Yang, Xiaojie; Gu, Qilin; Lin, Li; Li, Shaoyang; Zhong, Shan

    2015-01-01

    Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/β-catenin pathway positively modulates nup62l transcription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulate nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitation of nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish. PMID:25605329

  10. PPARα-UGT axis activation represses intestinal FXR-FGF15 feedback signalling and exacerbates experimental colitis

    PubMed Central

    Zhou, Xueyan; Cao, Lijuan; Jiang, Changtao; Xie, Yang; Cheng, Xuefang; Krausz, Kristopher W.; Qi, Yunpeng; Sun, Lu; Shah, Yatrik M.; Gonzalez, Frank J.; Wang, Guangji; Hao, Haiping

    2014-01-01

    Bile acids play a pivotal role in the pathological development of inflammatory bowel disease (IBD). However, the mechanism of bile acid dysregulation in IBD remains unanswered. Here we show that intestinal peroxisome proliferator-activated receptor α (PPARα)-UDP-glucuronosyltransferases (UGTs) signalling is an important determinant of bile acid homeostasis. Dextran sulphate sodium (DSS)-induced colitis leads to accumulation of bile acids in inflamed colon tissues via activation of the intestinal peroxisome PPARα-UGTs pathway. UGTs accelerate the metabolic elimination of bile acids, and thereby decrease their intracellular levels in the small intestine. Reduced intracellular bile acids results in repressed farnesoid X receptor (FXR)-FGF15 signalling, leading to upregulation of hepatic CYP7A1, thus promoting the de novo bile acid synthesis. Both knockout of PPARα and treatment with recombinant FGF19 markedly attenuate DSS-induced colitis. Thus, we propose that intestinal PPARα-UGTs and downstream FXR-FGF15 signalling play vital roles in control of bile acid homeostasis and the pathological development of colitis. PMID:25183423

  11. Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids*

    PubMed Central

    Anavi-Goffer, Sharon; Baillie, Gemma; Irving, Andrew J.; Gertsch, Jürg; Greig, Iain R.; Pertwee, Roger G.; Ross, Ruth A.

    2012-01-01

    GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55. PMID:22027819

  12. The apelin receptor: Physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR

    PubMed Central

    Chapman, Nigel A.; Dupré, Denis J.; Rainey, Jan K.

    2016-01-01

    The apelin receptor (AR or APJ) is a class A (rhodopsin-like) G-protein coupled receptor (GPCR) with wide distribution throughout the human body. Activation of the AR by its cognate peptide ligand, apelin, induces diverse physiological effects including vasoconstriction and dilation; strengthening of heart muscle contractility; angiogenesis; and, regulation of energy metabolism and fluid homeostasis. Recently, another endogenous peptidic activator of the AR, Toddler/ELABELA, was identified as having a crucial role in zebrafish embryonic development. The AR is also implicated in pathologies including cardiovascular disease, diabetes, obesity and cancer, making it a promising therapeutic target. Despite its established importance, the precise roles of AR signalling remain poorly understood. Moreover, little is known about mechanisms of peptide-AR activation. Additional complexity arises from modulation of the AR by two endogenous peptide ligands, both with multiple bioactive isoforms of variable length and distribution. The various apelin and Toddler/ELABELA isoforms may also produce distinct cellular effects. Further complexity arises through formation of functionally distinct heterodimers between the AR and other GPCRs. This minireview outlines key (patho)physiological actions of the AR, addresses what is known about signal transduction downstream of AR activation, and concludes by discussing unique properties of the endogenous peptidic ligands of the AR. PMID:25275559

  13. Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo.

    PubMed

    Nagai, Taku; Nakamuta, Shinichi; Kuroda, Keisuke; Nakauchi, Sakura; Nishioka, Tomoki; Takano, Tetsuya; Zhang, Xinjian; Tsuboi, Daisuke; Funahashi, Yasuhiro; Nakano, Takashi; Yoshimoto, Junichiro; Kobayashi, Kenta; Uchigashima, Motokazu; Watanabe, Masahiko; Miura, Masami; Nishi, Akinori; Kobayashi, Kazuto; Yamada, Kiyofumi; Amano, Mutsuki; Kaibuchi, Kozo

    2016-02-01

    Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors. PMID:26804993

  14. Hep88 mAb-mediated paraptosis-like apoptosis in HepG2 cells via downstream upregulation and activation of caspase-3, caspase-8 and caspase-9.

    PubMed

    Mitupatum, Thantip; Aree, Kalaya; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Puthong, Songchan; Kangsadalampai, Sasichai; Rojpibulstit, Panadda

    2015-01-01

    Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future. PMID:25773824

  15. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    PubMed Central

    Li, Chen-Shuang; Zheng, Zhong; Su, Xiao-Xia; Wang, Fei; Ling, Michelle; Zou, Min; Zhou, Hong

    2016-01-01

    Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering. PMID:26989682

  16. RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

    PubMed Central

    Tam, See-Ying; Lilla, Jennifer N.; Chen, Ching-Cheng; Kalesnikoff, Janet; Tsai, Mindy

    2015-01-01

    Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells. PMID:26588713

  17. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  18. Canine adenovirus downstream processing protocol.

    PubMed

    Puig, Meritxell; Piedra, Jose; Miravet, Susana; Segura, María Mercedes

    2014-01-01

    Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %. PMID:24132487

  19. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    PubMed

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  20. Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion.

    PubMed

    Tervonen, T A; Belitškin, D; Pant, S M; Englund, J I; Marques, E; Ala-Hongisto, H; Nevalaita, L; Sihto, H; Heikkilä, P; Leidenius, M; Hewitson, K; Ramachandra, M; Moilanen, A; Joensuu, H; Kovanen, P E; Poso, A; Klefström, J

    2016-04-01

    Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination. PMID:26165838

  1. Rosiglitazone activation of PPARγ-dependent signaling is neuroprotective in mutant huntingtin expressing cells.

    PubMed

    Chiang, Ming-Chang; Cheng, Yi-Chuan; Nicol, Christopher J; Lin, Kuan-Hung; Yen, Chia-Hui; Chen, Shiang-Jiuun; Huang, Rong-Nan

    2015-11-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a crucial transcription factor for neuroprotection in several brain diseases. Using a mouse model of Huntington's Disease (HD), we recently showed that PPARγ not only played a major function in preventing HD, but also oral intake of a PPARγ agonist (thiazolidinedione, TZD) significantly reduced the formation of mutant Huntingtin (mHtt) aggregates in the brain (e.g., cortex and striatum). The molecular mechanisms by which PPARγ exerts its HD neuroprotective effects remain unresolved. We investigated whether the PPARγ agonist (rosiglitazone) mediates neuroprotection in the mHtt expressing neuroblastoma cell line (N2A). Here we show that rosiglitazone upregulated the endogenous expression of PPARγ, its downstream target genes (including PGC1α, NRF-1 and Tfam) and mitochondrial function in mHtt expressing N2A cells. Rosiglitazone treatment also significantly reduced mHtt aggregates that included ubiquitin (Ub) and heat shock factor 1 (HSF1), as assessed by a filter-retardation assay, and increased the levels of the functional ubiquitin-proteasome system (UPS), HSF1 and heat shock protein 27/70 (HSP27/70) in N2A cells. Moreover, rosiglitazone treatment normalized endoplasmic reticulum (ER) stress sensors Bip, CHOP and ASK1, and significantly increased N2A cell survival. Taken together, these findings unveil new insights into the mechanisms by which activation of PPARγ signaling protects against the HD-mediated neuronal impairment. Further, our data also support the concept that PPARγ may be a novel therapeutic target for treating HD. PMID:26362846

  2. Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity.

    PubMed

    Lambert, Nevin A; Johnston, Christopher A; Cappell, Steven D; Kuravi, Sudhakiranmayi; Kimple, Adam J; Willard, Francis S; Siderovski, David P

    2010-04-13

    G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context. PMID:20351284

  3. Key gravity-sensitive signaling pathways drive T cell activation.

    PubMed

    Boonyaratanakornkit, J B; Cogoli, A; Li, C-F; Schopper, T; Pippia, P; Galleri, G; Meloni, M A; Hughes-Fulford, M

    2005-12-01

    Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity. PMID:16210397

  4. The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity.

    PubMed

    Wicher, Dieter; Derst, Christian; Gautier, Hélène; Lapied, Bruno; Heinemann, Stefan H; Agricola, Hans-Jürgen

    2007-01-01

    The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC(50)=11pM) due to reduction of a pacemaker Ca(2+) current through cAMP-inhibited pTRPgamma channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca(2+) concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPgamma channel that is activated by AKH under conditions of food shortage. PMID:18946521

  5. The Satiety Signaling Neuropeptide Perisulfakinin Inhibits the Activity of Central Neurons Promoting General Activity

    PubMed Central

    Wicher, Dieter; Derst, Christian; Gautier, Hélène; Lapied, Bruno; Heinemann, Stefan H.; Agricola, Hans-Jürgen

    2007-01-01

    The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM) due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage. PMID:18946521

  6. Deriving stellar inclination of slow rotators using stellar activity signal

    NASA Astrophysics Data System (ADS)

    Dumusque, Xavier

    2015-01-01

    Stellar inclination is an important parameter for many astrophysical studies. In the context of exoplanets, this allows us to derive the true obliquity of a system if the projected stellar spin-planetary orbit angle can measured via the Rossiter-Mclaughlin effect. Although different techniques allow us to estimate stellar inclination for fast rotators, it becomes much more difficult when stars are rotating slower than 2-2.5 km.s-1. By using the new activity simulation SOAP 2.0 that can reproduce the photometric and spectroscopic variations induced by stellar activity, we are able to fit the activity variation of solar-type stars and derive their inclination. The case of the equator-on star HD189733 will be presented, as well as the case of Alpha Centauri B, which present an inclination of 45+9-19 degrees, implying that the earth-mass orbiting planet is not transiting if aligned with its host star. Other exemples will also demonstrate the power of the technique, that can infer a stellar inclination, even for slow rotators like Alpha Centauri B, that present a projected rotational velocity smaller than 1.15 km.s-1. In addition, the SOAP 2.0 simulation can be used to correct for the effect of activity when one major active region is dominating the RV signal. This could enhance the detection of small mass exoplanets orbiting slightly active stars.This project is funded by ETAEARTH (European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement n. 313014), a transnational collaboration between European countries and the US (the Swiss Space Office, the Harvard Origin of Life Initiative, the Scottish Universities Physics Alliance, the University of Geneva, the Smithsonian Astrophysical Observatory, the Italian National Astrophysical Institute, the University of St. Andrews, Queens University Belfast, and the University of Edinburgh) setup to optimize the synergy between space-and ground-based data whose scientific potential for the characterization of

  7. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  8. Biomechanical Signals Inhibit IKK Activity to Attenuate NF-κB Transcription Activity in Inflamed Chondrocytes

    PubMed Central

    Dossumbekova, Anar; Anghelina, Mirela; Madhavan, Shashi; He, Lingli; Quan, Ning; Knobloch, Thomas; Agarwal, Sudha

    2016-01-01

    Objective While the effects of biomechanical signals in the form of joint movement and exercise are known to be beneficial to inflamed joints, limited information is available regarding the intracellular mechanisms of their actions. This study was undertaken to examine the intracellular mechanisms by which biomechanical signals suppress proinflammatory gene induction by the interleukin-1-β (IL-1β)–induced NF-κB signaling cascade in articular chondrocytes. Methods Primary rat articular chondrocytes were exposed to biomechanical signals in the form of cyclic tensile strain, and the effects on the NF-κB signaling cascade were examined by Western blot analysis, real-time polymerase chain reaction, and immunofluorescence. Results Cyclic tensile strain rapidly inhibited the IL-1β–induced nuclear translocation of NF-κB, but not its IL-1β–induced phosphorylation at serine 276 and serine 536, which are necessary for its transactivation and transcriptional efficacy, respectively. Examination of upstream events revealed that cyclic tensile strain also inhibited the cytoplasmic protein degradation of IκBβ and IκBα, as well as repressed their gene transcription. Additionally, cyclic tensile strain induced a rapid nuclear translocation of IκBα to potentially prevent NF-κB binding to DNA. Furthermore, the inhibition of IL-1β–induced degradation of IκB by cyclic tensile strain was mediated by down-regulation of IκB kinase activity. Conclusion These results indicate that the signals generated by cyclic tensile strain act at multiple sites within the NF-κB signaling cascade to inhibit IL-1β–induced proinflammatory gene induction. Taken together, these findings provide insight into how biomechanical signals regulate and reduce inflammation, and underscore their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints. PMID:17907174

  9. Gene Expression Patterns Associated with Peroxisome Proliferator-activated Receptor (PPAR) Signaling in the Longissimus dorsi of Hanwoo (Korean Cattle)

    PubMed Central

    Lim, Dajeong; Chai, Han-Ha; Lee, Seung-Hwan; Cho, Yong-Min; Choi, Jung-Woo; Kim, Nam-Kuk

    2015-01-01

    Adipose tissue deposited within muscle fibers, known as intramuscular fat (IMF or marbling), is a major determinant of meat quality and thereby affects its economic value. The biological mechanisms that determine IMF content are therefore of interest. In this study, 48 genes involved in the bovine peroxisome proliferator-activated receptor signaling pathway, which is involved in lipid metabolism, were investigated to identify candidate genes associated with IMF in the longissimus dorsi of Hanwoo (Korean cattle). Ten genes, retinoid X receptor alpha, peroxisome proliferator-activated receptor gamma (PPARG), phospholipid transfer protein, stearoyl-CoA desaturase, nuclear receptor subfamily 1 group H member 3, fatty acid binding protein 3 (FABP3), carnitine palmitoyltransferase II, acyl-Coenzyme A dehydrogenase long chain (ACADL), acyl-Coenzyme A oxidase 2 branched chain, and fatty acid binding protein 4, showed significant effects with regard to IMF and were differentially expressed between the low- and high-marbled groups (p<0.05). Analysis of the gene co-expression network based on Pearson’s correlation coefficients identified 10 up-regulated genes in the high-marbled group that formed a major cluster. Among these genes, the PPARG-FABP4 gene pair exhibited the strongest correlation in the network. Glycerol kinase was found to play a role in mediating activation of the differentially expressed genes. We categorized the 10 significantly differentially expressed genes into the corresponding downstream pathways and investigated the direct interactive relationships among these genes. We suggest that fatty acid oxidation is the major downstream pathway affecting IMF content. The PPARG/RXRA complex triggers activation of target genes involved in fatty acid oxidation resulting in increased triglyceride formation by ATP production. Our findings highlight candidate genes associated with the IMF content of the loin muscle of Korean cattle and provide insight into the

  10. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    PubMed Central

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  11. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells.

    PubMed

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as "S-mercuration", potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  12. Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

    PubMed Central

    Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

    2015-01-01

    Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H+-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity. PMID:26259639

  13. Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgamma2 and A2.

    PubMed

    Dellas, Claudia; Schäfer, Katrin; Rohm, Ilonka K; Lankeit, Mareike; Leifheit, Maren; Loskutoff, David J; Hasenfuss, Gerd; Konstantinides, Stavros V

    2007-11-01

    Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of alpha(IIb)beta(3), as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cgamma2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A(2) axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity. PMID:18000612

  14. α-Melanocyte stimulating hormone attenuates dexamethasone-induced osteoblast damages through activating melanocortin receptor 4-SphK1 signaling.

    PubMed

    Guo, Shiguang; Xie, Yue; Fan, Jian-bo; Ji, Feng; Wang, Shouguo; Fei, Haodong

    2016-01-01

    Long-term glucocorticoid (GC) usage may cause non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) is shown to exert potent cytotoxic effect to osteoblasts. Here, we investigated the potential activity of α-melanocyte stimulating hormone (α-MSH) against the process. Our data revealed that pretreatment of α-MSH significantly inhibited Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Melanocortin receptor 4 (MC4R) acts as the receptor of α-MSH in mediating its actions in osteoblasts. The MC4R antagonist SHU9119, or shRNA-mediated knockdown of MC4R, almost abolished α-MSH-induced activation of downstream signalings (Akt and Erk1/2) and its pro-survival effect in osteoblasts. Further studies showed that α-MSH activated MC4R downstream sphingosine kinase 1 (SphK1) and increased cellular sphingosine-1-phosphate (S1P) content in MC3T3-E1 cells and primary murine osteoblasts, which were blocked by SHU9119 or MC4R shRNAs. SphK1 inhibition by the its inhibitor N,N-dimethylsphingosine (DMS), or SphK1 knockdown by targeted-shRNAs, largely attenuated α-MSH-mediated osteoblast protection against Dex. Together, these results suggest that α-MSH alleviates Dex-induced damages to cultured osteoblasts through activating MC4R-SphK1 signaling. PMID:26631960

  15. Transcription of T cell receptor beta-chain genes is controlled by a downstream regulatory element.

    PubMed Central

    Krimpenfort, P; de Jong, R; Uematsu, Y; Dembic, Z; Ryser, S; von Boehmer, H; Steinmetz, M; Berns, A

    1988-01-01

    To characterize cis-acting elements controlling the expression of T cell receptor beta-chains we generated a number of transgenic mouse lines harboring a rearranged T cell receptor beta-chain with different extensions of 5' and 3' flanking sequences. Transcriptional analysis of transgenic mice carrying these clones showed that sequences located downstream of the polyadenylation signal of the C beta 2 region are indispensable for expression in transgenic mice. The sequences conferring enhancer activity in this fragment were further defined by transient CAT assays. Strong enhancer activity was found to reside in a 550 bp fragment located 5 kb downstream from C beta 2. The nucleotide sequence of this fragment revealed a number of oligonucleotide motifs characteristic for enhancer elements. Images PMID:3396541

  16. Regulation of MnSOD Enzymatic Activity by Sirt3 Connects the Mitochondrial Acetylome Signaling Networks to Aging and Carcinogenesis

    PubMed Central

    Tao, Randa; Vassilopoulos, Athanassios; Parisiadou, Loukia; Yan, Yufan

    2014-01-01

    Abstract Significance: It is a well-established scientific observation that mammalian cells contain fidelity or watchdog proteins that maintain the correct function of cellular organelles. Recent Advances: Over the past several years, the Sirtuin deacetylase family protein Sirt3 has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species (ROS) scavenging proteins. Loss of function or genetic mutation of these fidelity proteins has been shown to create a cellular environment that is permissive for the development of cellular damage associated with processes such as aging and carcinogenesis. Critical Issues: Mitochondria are the primary organelles that direct oxidative metabolism for the production of ATP; however, this is also a significant source of ROS. Thus, it is reasonable to propose that mitochondria should contain proteins that would signal downstream target molecules and/or ROS scavenger enzymes to maintain mitochondrial and cellular homeostatic poise. It is also reasonable to hypothesize that the mitochondria contain fidelity proteins similar to those found in the nucleus and cytoplasm. We discuss a new role of Sirt3 in the direction of the primary superoxide scavenger protein, manganese superoxide dismutase (MnSOD), and how the acetylation or deacetylation of several specific lysines appears to direct MnSOD enzymatic dismutase activity. Future Directions: Aberrant downstream regulation of MnSOD by Sirt3 may be a potential source of cellular damage that accumulates with aging to create a tumor-permissive phenotype. Future studies can explore the role of MnSOD in age-related illness using this new mechanism of enzymatic regulation. Antioxid. Redox Signal. 20, 1646–1654 PMID:23886445