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Sample records for 1b mediates signaling

  1. Dysregulated YAP1/TAZ and TGF-β signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice.

    PubMed

    Nishio, Miki; Sugimachi, Keishi; Goto, Hiroki; Wang, Jia; Morikawa, Takumi; Miyachi, Yosuke; Takano, Yusuke; Hikasa, Hiroki; Itoh, Tohru; Suzuki, Satoshi O; Kurihara, Hiroki; Aishima, Shinichi; Leask, Andrew; Sasaki, Takehiko; Nakano, Toru; Nishina, Hiroshi; Nishikawa, Yuji; Sekido, Yoshitaka; Nakao, Kazuwa; Shin-Ya, Kazuo; Mimori, Koshi; Suzuki, Akira

    2016-01-01

    Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial-mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-β)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-β pathway may be effective treatment for cHC-CCs and ICCs.

  2. Dysregulated YAP1/TAZ and TGF-β signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice

    PubMed Central

    Nishio, Miki; Sugimachi, Keishi; Goto, Hiroki; Wang, Jia; Morikawa, Takumi; Miyachi, Yosuke; Takano, Yusuke; Hikasa, Hiroki; Itoh, Tohru; Suzuki, Satoshi O.; Kurihara, Hiroki; Aishima, Shinichi; Leask, Andrew; Sasaki, Takehiko; Nakano, Toru; Nishina, Hiroshi; Nishikawa, Yuji; Sekido, Yoshitaka; Nakao, Kazuwa; Shin-ya, Kazuo; Mimori, Koshi; Suzuki, Akira

    2016-01-01

    Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial–mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-β)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-β pathway may be effective treatment for cHC-CCs and ICCs. PMID:26699479

  3. FSH-induced p38-MAPK-mediated dephosphorylation at serine 727 of the signal transducer and activator of transcription 1 decreases Cyp1b1 expression in mouse granulosa cells.

    PubMed

    Du, Xue-Hai; Zhou, Xiao-Long; Cao, Rui; Xiao, Peng; Teng, Yun; Ning, Cai-Bo; Liu, Hong-Lin

    2015-01-01

    Most mammalian follicles undergo atresia at various stages before ovulation, and granulosa cell apoptosis is a major cause of antral follicular atresia. Estradiol is an essential mitogen for granulosa cell proliferation in vivo and inhibition of apoptosis. The estradiol-producing capacity and metabolism levels are important for follicle health, and sufficient estradiol is necessary for follicle development and ovulation. Cyp1b1, a member of the cytochrome P450 1 subfamily, is responsible for the metabolism of a wide variety of halogenated and polycyclic aromatic hydrocarbons in diverse tissues. In mouse follicles, Cyp1b1 converts estradiol to 4-hydroxyestradiol. We investigated mouse granulosa cells (MGCs) in vivo and in vitro and found that Cyp1b1 played a crucial role in estradiol metabolism in dominant follicles. Follicle-stimulating hormone (FSH) decreased estrogen metabolism by reducing Cyp1b1 mRNA and protein levels in MGCs. Furthermore, FSH regulated signal transducer and activator of transcription 1 (STAT1), a significant transcription factor of Cyp1b1, by mediating the dephosphorylation of STAT1 on serine 727 (Ser(727)) in MGCs. p38 mitogen-activated protein kinase (MAPK) may be involved in the FSH-induced dephosphorylation of STAT1 on Ser(727) in MGCs. These results suggested that FSH functions via p38 MAPK-induced dephosphorylation at Ser(727) of STAT1 to downregulate Cyp1b1 expression and maintain the estradiol levels in mouse dominant follicles.

  4. Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3

    PubMed Central

    Gui, Chunshan; Miao, Yi; Thompson, Lucas; Wahlgren, Bret; Mock, Melissa; Stieger, Bruno; Hagenbuch, Bruno

    2008-01-01

    The pregnane X receptor is a ligand-activated transcription factor that is abundantly expressed in hepatocytes. Numerous drugs are pregnane X receptor ligands. To bind to their receptor they must cross the sinusoidal membrane. Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are polyspecific transporters expressed at the sinusoidal membrane of human hepatocytes. They mediate transport of a variety of drugs including the pregnane X receptor ligands rifampicin and dexamethasone. To test whether additional pregnane X receptor ligands interact with OATP1B1- and 1B3-mediated transport, we developed Chinese Hamster Ovary (CHO) cell lines stably expressing OATP1B1 or 1B3 at high levels. OATP1B1- and 1B3-mediated estradiol-17β-glucuronide uptake was inhibited by several pregnane X receptor ligands in a concentration dependent way. IC50 values for rifampicin, paclitaxel, mifepristone, and troglitazone were within their respective pharmacological free plasma concentrations. Kinetic analysis revealed that clotrimazole inhibits OATP1B1-mediated estradiol-17β-glucuronide transport with a Ki of 7.7 ± 0.3 μM in a competitive way. However, uptake of OATP1B3-mediated estradiol-17β-glucuronide was stimulated and this stimulation was due to an increased apparent affinity. Transport of estrone-3-sulfate was hardly affected while all other substrates tested were inhibited. Additional azoles like fluconazole, ketoconazole and miconazole did not stimulate OATP1B3-mediated estradiol-17β-glucuronide transport. In summary, these results demonstrate that pregnane X receptor ligands, by inhibiting or stimulating OATP-mediated uptake, can lead to drug-drug interactions at the transporter level. PMID:18321482

  5. Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling

    PubMed Central

    Vasileiou, Georgia; Ekici, Arif B.; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V.

    2015-01-01

    The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. PMID:26340334

  6. Essential role of protein-tyrosine phosphatase 1B in the modulation of insulin signaling by acetaminophen in hepatocytes.

    PubMed

    Mobasher, Maysa Ahmed; de Toro-Martín, Juan; González-Rodríguez, Águeda; Ramos, Sonia; Letzig, Lynda G; James, Laura P; Muntané, Jordi; Álvarez, Carmen; Valverde, Ángela M

    2014-10-17

    Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B(-/-) mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver. PMID:25204659

  7. Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes*

    PubMed Central

    Mobasher, Maysa Ahmed; de Toro-Martín, Juan; González-Rodríguez, Águeda; Ramos, Sonia; Letzig, Lynda G.; James, Laura P.; Muntané, Jordi; Álvarez, Carmen; Valverde, Ángela M.

    2014-01-01

    Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B−/− mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver. PMID:25204659

  8. Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide

    PubMed Central

    Jeong, Hyeon-Uk; Kwon, Mihwa; Lee, Yongnam; Yoo, Ji Seok; Shin, Dae Hee; Song, Im-Sook; Lee, Hye Suk

    2015-01-01

    We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (Km) =41.5 μM, maximum uptake rate (Vmax) =46.2 pmol/minute, and intrinsic clearance (CLint) =1.11 μL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CLint values of 0.035 and 0.034 μL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 μM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb–drug interactions of catalposide, although their clinical relevance awaits further evaluation. PMID:25653502

  9. Pharmacokinetic effects of curcumin on docetaxel mediated by OATP1B1, OATP1B3 and CYP450s.

    PubMed

    Sun, Xiaolin; Li, Junxiu; Guo, Chaorui; Xing, Han; Xu, Jie; Wen, Yanli; Qiu, Zhixia; Zhang, Qiuyang; Zheng, Yi; Chen, Xijing; Zhao, Di

    2016-08-01

    Curcumin can synergistically enhance docetaxel's in vitro and in vivo antitumor activity and has been co-administrated with docetaxel in clinical trials. The aim of our study is to investigate the effect of curcumin on the pharmacokinetics of docetaxel and explore its mechanism on OATP1B1, OATP1B3 and human liver microsomes (HLMs). In rats, curcumin increased the docetaxel area under the plasma concentration-time curve (AUC0-8h) and the terminal half-life (t1/2) to 1.86- and 1.55-fold, respectively. Moreover, curcumin decreased the clearance (CL) of docetaxel to 52.1%. Human embryonic kidney 293 (HEK293) cells stably expressing OATP1B1 and OATP1B3 were used to observe the effects of curcumin on OATP1B1 and OATP1B3-mediated uptake of docetaxel. Curcumin exhibited potent inhibition on OATP1B1 and OATP1B3-mediated docetaxel uptake with IC50 values of 3.81 ± 1.19 μM and 33.70 ± 1.22 μM, respectively. The inhibition of curcumin on docetaxel metabolism in HLMs indicated that curcumin can modestly inhibit the metabolism of docetaxel with the IC50 value of 22.70 ± 1.13 μM and Ki value of 24.72 ± 4.24 μM. The preclinical and clinical improved docetaxel's therapeutic efficacy when co-administrated with curcumin may be due to the inhibition of curcumin on OATP1B1, OATP1B3 and HLMs activities. Close attention should be paid when combined treatment with docetaxel and curcumin carried out clinically. PMID:27452633

  10. Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk

    PubMed Central

    Painter, Jodie N.; O'Mara, Tracy A.; Batra, Jyotsna; Cheng, Timothy; Lose, Felicity A.; Dennis, Joe; Michailidou, Kyriaki; Tyrer, Jonathan P.; Ahmed, Shahana; Ferguson, Kaltin; Healey, Catherine S.; Kaufmann, Susanne; Hillman, Kristine M.; Walpole, Carina; Moya, Leire; Pollock, Pamela; Jones, Angela; Howarth, Kimberley; Martin, Lynn; Gorman, Maggie; Hodgson, Shirley; De Polanco, Ma. Magdalena Echeverry; Sans, Monica; Carracedo, Angel; Castellvi-Bel, Sergi; Rojas-Martinez, Augusto; Santos, Erika; Teixeira, Manuel R.; Carvajal-Carmona, Luis; Shu, Xiao-Ou; Long, Jirong; Zheng, Wei; Xiang, Yong-Bing; Montgomery, Grant W.; Webb, Penelope M.; Scott, Rodney J.; McEvoy, Mark; Attia, John; Holliday, Elizabeth; Martin, Nicholas G.; Nyholt, Dale R.; Henders, Anjali K.; Fasching, Peter A.; Hein, Alexander; Beckmann, Matthias W.; Renner, Stefan P.; Dörk, Thilo; Hillemanns, Peter; Dürst, Matthias; Runnebaum, Ingo; Lambrechts, Diether; Coenegrachts, Lieve; Schrauwen, Stefanie; Amant, Frederic; Winterhoff, Boris; Dowdy, Sean C.; Goode, Ellen L.; Teoman, Attila; Salvesen, Helga B.; Trovik, Jone; Njolstad, Tormund S.; Werner, Henrica M.J.; Ashton, Katie; Proietto, Tony; Otton, Geoffrey; Tzortzatos, Gerasimos; Mints, Miriam; Tham, Emma; Hall, Per; Czene, Kamila; Liu, Jianjun; Li, Jingmei; Hopper, John L.; Southey, Melissa C.; Ekici, Arif B.; Ruebner, Matthias; Johnson, Nicola; Peto, Julian; Burwinkel, Barbara; Marme, Frederik; Brenner, Hermann; Dieffenbach, Aida K.; Meindl, Alfons; Brauch, Hiltrud; Lindblom, Annika; Depreeuw, Jeroen; Moisse, Matthieu; Chang-Claude, Jenny; Rudolph, Anja; Couch, Fergus J.; Olson, Janet E.; Giles, Graham G.; Bruinsma, Fiona; Cunningham, Julie M.; Fridley, Brooke L.; Børresen-Dale, Anne-Lise; Kristensen, Vessela N.; Cox, Angela; Swerdlow, Anthony J.; Orr, Nicholas; Bolla, Manjeet K.; Wang, Qin; Weber, Rachel Palmieri; Chen, Zhihua; Shah, Mitul; French, Juliet D.; Pharoah, Paul D.P.; Dunning, Alison M.; Tomlinson, Ian; Easton, Douglas F.; Edwards, Stacey L.; Thompson, Deborah J.; Spurdle, Amanda B.

    2015-01-01

    Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10−14, odds ratio = 0.86, 95% confidence interval = 0.82–0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression. PMID:25378557

  11. Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk.

    PubMed

    Painter, Jodie N; O'Mara, Tracy A; Batra, Jyotsna; Cheng, Timothy; Lose, Felicity A; Dennis, Joe; Michailidou, Kyriaki; Tyrer, Jonathan P; Ahmed, Shahana; Ferguson, Kaltin; Healey, Catherine S; Kaufmann, Susanne; Hillman, Kristine M; Walpole, Carina; Moya, Leire; Pollock, Pamela; Jones, Angela; Howarth, Kimberley; Martin, Lynn; Gorman, Maggie; Hodgson, Shirley; De Polanco, Ma Magdalena Echeverry; Sans, Monica; Carracedo, Angel; Castellvi-Bel, Sergi; Rojas-Martinez, Augusto; Santos, Erika; Teixeira, Manuel R; Carvajal-Carmona, Luis; Shu, Xiao-Ou; Long, Jirong; Zheng, Wei; Xiang, Yong-Bing; Montgomery, Grant W; Webb, Penelope M; Scott, Rodney J; McEvoy, Mark; Attia, John; Holliday, Elizabeth; Martin, Nicholas G; Nyholt, Dale R; Henders, Anjali K; Fasching, Peter A; Hein, Alexander; Beckmann, Matthias W; Renner, Stefan P; Dörk, Thilo; Hillemanns, Peter; Dürst, Matthias; Runnebaum, Ingo; Lambrechts, Diether; Coenegrachts, Lieve; Schrauwen, Stefanie; Amant, Frederic; Winterhoff, Boris; Dowdy, Sean C; Goode, Ellen L; Teoman, Attila; Salvesen, Helga B; Trovik, Jone; Njolstad, Tormund S; Werner, Henrica M J; Ashton, Katie; Proietto, Tony; Otton, Geoffrey; Tzortzatos, Gerasimos; Mints, Miriam; Tham, Emma; Hall, Per; Czene, Kamila; Liu, Jianjun; Li, Jingmei; Hopper, John L; Southey, Melissa C; Ekici, Arif B; Ruebner, Matthias; Johnson, Nicola; Peto, Julian; Burwinkel, Barbara; Marme, Frederik; Brenner, Hermann; Dieffenbach, Aida K; Meindl, Alfons; Brauch, Hiltrud; Lindblom, Annika; Depreeuw, Jeroen; Moisse, Matthieu; Chang-Claude, Jenny; Rudolph, Anja; Couch, Fergus J; Olson, Janet E; Giles, Graham G; Bruinsma, Fiona; Cunningham, Julie M; Fridley, Brooke L; Børresen-Dale, Anne-Lise; Kristensen, Vessela N; Cox, Angela; Swerdlow, Anthony J; Orr, Nicholas; Bolla, Manjeet K; Wang, Qin; Weber, Rachel Palmieri; Chen, Zhihua; Shah, Mitul; French, Juliet D; Pharoah, Paul D P; Dunning, Alison M; Tomlinson, Ian; Easton, Douglas F; Edwards, Stacey L; Thompson, Deborah J; Spurdle, Amanda B

    2015-03-01

    Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression.

  12. Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk.

    PubMed

    Painter, Jodie N; O'Mara, Tracy A; Batra, Jyotsna; Cheng, Timothy; Lose, Felicity A; Dennis, Joe; Michailidou, Kyriaki; Tyrer, Jonathan P; Ahmed, Shahana; Ferguson, Kaltin; Healey, Catherine S; Kaufmann, Susanne; Hillman, Kristine M; Walpole, Carina; Moya, Leire; Pollock, Pamela; Jones, Angela; Howarth, Kimberley; Martin, Lynn; Gorman, Maggie; Hodgson, Shirley; De Polanco, Ma Magdalena Echeverry; Sans, Monica; Carracedo, Angel; Castellvi-Bel, Sergi; Rojas-Martinez, Augusto; Santos, Erika; Teixeira, Manuel R; Carvajal-Carmona, Luis; Shu, Xiao-Ou; Long, Jirong; Zheng, Wei; Xiang, Yong-Bing; Montgomery, Grant W; Webb, Penelope M; Scott, Rodney J; McEvoy, Mark; Attia, John; Holliday, Elizabeth; Martin, Nicholas G; Nyholt, Dale R; Henders, Anjali K; Fasching, Peter A; Hein, Alexander; Beckmann, Matthias W; Renner, Stefan P; Dörk, Thilo; Hillemanns, Peter; Dürst, Matthias; Runnebaum, Ingo; Lambrechts, Diether; Coenegrachts, Lieve; Schrauwen, Stefanie; Amant, Frederic; Winterhoff, Boris; Dowdy, Sean C; Goode, Ellen L; Teoman, Attila; Salvesen, Helga B; Trovik, Jone; Njolstad, Tormund S; Werner, Henrica M J; Ashton, Katie; Proietto, Tony; Otton, Geoffrey; Tzortzatos, Gerasimos; Mints, Miriam; Tham, Emma; Hall, Per; Czene, Kamila; Liu, Jianjun; Li, Jingmei; Hopper, John L; Southey, Melissa C; Ekici, Arif B; Ruebner, Matthias; Johnson, Nicola; Peto, Julian; Burwinkel, Barbara; Marme, Frederik; Brenner, Hermann; Dieffenbach, Aida K; Meindl, Alfons; Brauch, Hiltrud; Lindblom, Annika; Depreeuw, Jeroen; Moisse, Matthieu; Chang-Claude, Jenny; Rudolph, Anja; Couch, Fergus J; Olson, Janet E; Giles, Graham G; Bruinsma, Fiona; Cunningham, Julie M; Fridley, Brooke L; Børresen-Dale, Anne-Lise; Kristensen, Vessela N; Cox, Angela; Swerdlow, Anthony J; Orr, Nicholas; Bolla, Manjeet K; Wang, Qin; Weber, Rachel Palmieri; Chen, Zhihua; Shah, Mitul; French, Juliet D; Pharoah, Paul D P; Dunning, Alison M; Tomlinson, Ian; Easton, Douglas F; Edwards, Stacey L; Thompson, Deborah J; Spurdle, Amanda B

    2015-03-01

    Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression. PMID:25378557

  13. Myosin 1b functions as an effector of EphB signaling to control cell repulsion

    PubMed Central

    Prospéri, Marie-Thérèse; Lépine, Priscilla; Dingli, Florent; Paul-Gilloteaux, Perrine; Martin, René; Loew, Damarys; Knölker, Hans-Joachim

    2015-01-01

    Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion. PMID:26195670

  14. Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease.

    PubMed

    Yang, Chiao-Wen; Hojer, Caroline D; Zhou, Meijuan; Wu, Xiumin; Wuster, Arthur; Lee, Wyne P; Yaspan, Brian L; Chan, Andrew C

    2016-01-14

    The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

  15. Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

    PubMed Central

    2013-01-01

    It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. PMID:23442260

  16. The arginine vasopressin V1b receptor gene and prosociality: Mediation role of emotional empathy.

    PubMed

    Wu, Nan; Shang, Siyuan; Su, Yanjie

    2015-09-01

    The vasopressin V1b receptor (AVPR1B) gene has been shown to be closely associated with bipolar disorder and depression. However, whether it relates to positive social outcomes, such as empathy and prosocial behavior, remains unknown. This study explored the possible role of the AVPR1B gene rs28373064 in empathy and prosociality. A total of 256 men, who were genetically unrelated, non-clinical ethnic Han Chinese college students, participated in the study. Prosociality was tested by measuring the prosocial tendencies of cognitive and emotional empathy using the Interpersonal Reactivity Index (IRI). The single nucleotide polymorphism (SNP), rs28373064, was genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results suggest that the AVPR1B gene rs28373064 is linked to emotional empathy and prosociality. The mediation analysis indicated that the effect of the AVPR1B gene on prosociality might be mediated by emotional empathy. This study demonstrated the link between the AVPR1B gene and prosociality and provided evidence that emotional empathy might mediate the relation between the AVPR1B gene and prosociality.

  17. Plectin isoform 1b mediates mitochondrion-intermediate filament network linkage and controls organelle shape.

    PubMed

    Winter, Lilli; Abrahamsberg, Christina; Wiche, Gerhard

    2008-06-16

    Plectin is a versatile intermediate filament (IF)-bound cytolinker protein with a variety of differentially spliced isoforms accounting for its multiple functions. One particular isoform, plectin 1b (P1b), remains associated with mitochondria after biochemical fractionation of fibroblasts and cells expressing exogenous P1b. Here, we determined that P1b is inserted into the outer mitochondrial membrane with the exon 1b-encoded N-terminal sequence serving as a mitochondrial targeting and anchoring signal. To study P1b-related mitochondrial functions, we generated mice that selectively lack this isoform but express all others. In primary fibroblasts and myoblasts derived from these mice, we observe a substantial elongation of mitochondrial networks, whereas other mitochondrial properties remain largely unaffected. Normal morphology of mitochondria could be restored by isoform-specific overexpression of P1b in P1b-deficient as well as plectin-null cells. We propose a model where P1b both forms a mitochondrial signaling platform and affects organelle shape and network formation by tethering mitochondria to IFs.

  18. CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation.

    PubMed

    Kwon, Yeo-Jung; Baek, Hyoung-Seok; Ye, Dong-Jin; Shin, Sangyun; Kim, Donghak; Chun, Young-Jin

    2016-01-01

    Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction. PMID:26981862

  19. CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation

    PubMed Central

    Kwon, Yeo-Jung; Baek, Hyoung-Seok; Ye, Dong-Jin; Shin, Sangyun; Kim, Donghak; Chun, Young-Jin

    2016-01-01

    Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction. PMID:26981862

  20. The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

    PubMed Central

    Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; González, Alfonso; Bonifacino, Juan S.

    2014-01-01

    SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface. PMID:24229647

  1. Influence of Drug Formulation on OATP1B-Mediated Transport of Paclitaxel

    PubMed Central

    Nieuweboer, Annemieke J.M.; Hu, Shuiying; Hagenbuch, Bruno; Moghaddam-Helmantel, Inge Ghobadi; Gibson, Alice A.; de Bruijn, Peter; Mathijssen, Ron H. J.; Sparreboom, Alex

    2014-01-01

    Purpose Taxane antineoplastic agents are extensively taken up into hepatocytes by OATP1B-type transporters prior to metabolism and excretion. Because the biodistributional properties imposed upon these agents by different solubilizers drive clinically-important pharmacodynamic endpoints, we tested the hypothesis that the in vitro and in vivo interaction of taxanes with OATP1B transporters is affected by the choice of drug delivery system. Experimental Design Transport of paclitaxel, docetaxel, and cabazitaxel was studied in vitro using various cell lines transfected with OATP1B1, OATP1B3, or the rodent equivalent Oatp1b2. Pharmacokinetic studies were done in wildtype and Oatp1b2-knockout mice in the presence or absence of polysorbate 80 (PS80) or Kolliphor EL (formerly Cremophor EL; CrEL). Results Paclitaxel and docetaxel, but not cabazitaxel, were transported substrates of OATP1B1, OATP1B3, and Oatp1b2, and these transport processes were strongly reduced in the presence of clinically-relevant concentrations of PS80 and CrEL. In the absence of solubilizers, deficiency of Oatp1b2 in mice was associated with a significantly decreased taxane clearance due to a liver distribution defect (P<0.00001), but these kinetic changes were masked in the presence of PS80 or CrEL (P>0.05). Conclusions Our findings confirm the importance of OATP1B-type transporters in the hepatic elimination of taxanes, and that this process can be inhibited by PS80 and CrEL. These results suggest that the likelihood of drug-drug interactions mediated by these transporters is strongly dependent on the selected taxane solubilizer. PMID:24755470

  2. Zebrafish cdx1b regulates expression of downstream factors of Nodal signaling during early endoderm formation.

    PubMed

    Cheng, Pei-Yi; Lin, Chia-Chi; Wu, Chun-Shiu; Lu, Yu-Fen; Lin, Che Yi; Chung, Chih-Ching; Chu, Cheng-Ying; Huang, Chang-Jen; Tsai, Chun-Yen; Korzh, Svetlana; Wu, Jen-Leih; Hwang, Sheng-Ping L

    2008-03-01

    We identified a zebrafish caudal-related homeobox (cdx1b) gene, which shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs. PMID:18234726

  3. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena

    PubMed Central

    Hughes, Shannon K.; Oudin, Madeleine J.; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A.; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S.; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A.; Gertler, Frank B.

    2015-01-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express MenaINV, which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5′ inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When MenaINV is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor–induced signaling. Disruption of this attenuation by MenaINV sensitizes tumor cells to low–growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. PMID:26337385

  4. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    PubMed

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.

  5. Deletion of protein tyrosine phosphatase 1b in proopiomelanocortin neurons reduces neurogenic control of blood pressure and protects mice from leptin- and sympatho-mediated hypertension.

    PubMed

    Bruder-Nascimento, Thiago; Butler, Benjamin R; Herren, David J; Brands, Michael W; Bence, Kendra K; Belin de Chantemèle, Eric J

    2015-12-01

    Protein tyrosine phosphatase 1b (Ptp1b), which represses leptin signaling, is a promising therapeutic target for obesity. Genome wide deletion of Ptp1b, increases leptin sensitivity, protects mice from obesity and diabetes, but alters cardiovascular function by increasing blood pressure (BP). Leptin-control of metabolism is centrally mediated and involves proopiomelanocortin (POMC) neurons. Whether these neurons contribute to leptin-mediated increases in BP remain unclear. We hypothesized that increasing leptin signaling in POMC neurons with Ptp1b deletion will sensitize the cardiovascular system to leptin and enhance neurogenic control of BP. We analyzed the cardiovascular phenotype of Ptp1b+/+ and POMC-Ptp1b-/- mice, at baseline and after 7 days of leptin infusion or sympatho-activation with phenylephrine. POMCPtp1b deletion did not alter baseline cardiovascular hemodynamics (BP, heart rate) but reduced BP response to ganglionic blockade and plasma catecholamine levels that suggests a decreased neurogenic control of BP. In contrast, POMC-Ptp1b deletion increased vascular adrenergic reactivity and aortic α-adrenergic receptors expression. Chronic leptin treatment reduced vascular adrenergic reactivity and blunted diastolic and mean BP increases in POMC-Ptp1b-/- mice only. Similarly POMC-Ptp1b-/- mice exhibited a blunted increased in diastolic and mean BP accompanied by a gradual reduction in adrenergic reactivity in response to chronic vascular sympatho-activation with phenylephrine. Together these data rule out our hypothesis but suggest that deletion of Ptp1b in POMC neurons protects from leptin- and sympatho-mediated increases in BP. Vascular adrenergic desensitization appears as a protective mechanism against hypertension, and POMC-Ptp1b as a key therapeutic target for the treatment of metabolic and cardiovascular dysfunctions associated with obesity.

  6. Cell autonomous regulation of hippocampal circuitry via Aph1b-γ-secretase/neuregulin 1 signalling

    PubMed Central

    Fazzari, Pietro; Snellinx, An; Sabanov, Victor; Ahmed, Tariq; Serneels, Lutgarde; Gartner, Annette; Shariati, S Ali M; Balschun, Detlef; De Strooper, Bart

    2014-01-01

    Neuregulin 1 (NRG1) and the γ-secretase subunit APH1B have been previously implicated as genetic risk factors for schizophrenia and schizophrenia relevant deficits have been observed in rodent models with loss of function mutations in either gene. Here we show that the Aph1b-γ-secretase is selectively involved in Nrg1 intracellular signalling. We found that Aph1b-deficient mice display a decrease in excitatory synaptic markers. Electrophysiological recordings show that Aph1b is required for excitatory synaptic transmission and plasticity. Furthermore, gain and loss of function and genetic rescue experiments indicate that Nrg1 intracellular signalling promotes dendritic spine formation downstream of Aph1b-γ-secretase in vitro and in vivo. In conclusion, our study sheds light on the physiological role of Aph1b-γ-secretase in brain and provides a new mechanistic perspective on the relevance of NRG1 processing in schizophrenia. DOI: http://dx.doi.org/10.7554/eLife.02196.001 PMID:24891237

  7. Snail Recruits Ring1B to Mediate Transcriptional Repression and Cell Migration in Pancreatic Cancer Cells

    PubMed Central

    Chen, Jiangzhi; Xu, Hong; Zou, Xiuqun; Wang, Jiamin; Zhu, Yi; Chen, Hao; Shen, Baiyong; Deng, Xiaxing; Zhou, Aiwu; Chin, Y. Eugene; Rauscher, Frank J.; Peng, Chenghong; Hou, Zhaoyuan

    2014-01-01

    Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 4353-63. ©2014 AACR PMID:24903147

  8. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    PubMed Central

    González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

    2016-01-01

    ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

  9. Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions

    PubMed Central

    Castillo-Badillo, Jean A.; Molina-Muñoz, Tzindilú; Romero-Ávila, M. Teresa; Vázquez-Macías, Aleida; Rivera, Richard; Chun, Jerold; García-Sáinz, J. Adolfo

    2012-01-01

    Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation was studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appears to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor- α1B-adrenergic receptor crosstalk. PMID:22019450

  10. 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

  11. Regulation of growth hormone induced JAK2 and mTOR signalling by hepatic protein tyrosine phosphatase 1B.

    PubMed

    Owen, C; Lees, E K; Mody, N; Delibegović, M

    2015-02-01

    Protein tyrosine phosphatase 1B (PTP1B) regulates various signalling pathways including insulin, leptin, IGF-1 and growth hormone (GH) signalling. Transmission of the GH signal depends on Janus kinase 2 (JAK2), which is how PTP1B is thought to modulate GH signalling in the liver, based on studies utilising global PTP1B knockout mice (Ptp1b(-/-)). Here, we investigated the liver-specific role of PTP1B in GH signalling, using liver-specific Ptp1b(-/-) mice (alb-crePtp1b(-/-)), under physiological (chow) or insulin resistant (high-fat diet [HFD]) feeding conditions. Body weight and adiposity were comparable between female alb-crePtp1b(-/-) and Ptp1b(fl/fl) control mice. On chow diet, under 48-hour fasting GH-resistant conditions, GH stimulation in vivo led to a robust stimulation of the JAK-STAT signalling pathway. Alb-crePtp1b(-/-) mice exhibited significantly higher GH-induced JAK2 phosphorylation and SOCS3 gene expression post-GH stimulation. However, STAT3, STAT5 and ERK1/2 phosphorylation and SOCS2 gene expression were similar between groups. Interestingly, GH-induced mTOR phosphorylation was significantly higher in alb-crePtp1b(-/-) mice 5-min post-GH stimulation compared to controls, revealing this part of the pathway under direct control of PTP1B. Under ad lib HFD-fed conditions, GH-induced STAT5 phosphorylation significantly increased in alb-crePtp1b(-/-) mice only, with no alterations in the controls. Overall, our data demonstrate that liver-specific PTP1B deletion leads to significant alterations in GH signalling with increased JAK2, STAT5 and mTOR phosphorylation and SOCS3 gene expression.

  12. Divergence and Conservation of the Major UPR Branch IRE1-bZIP Signaling Pathway across Eukaryotes.

    PubMed

    Zhang, Lingrui; Zhang, Changwei; Wang, Aiming

    2016-01-01

    The unfolded protein response (UPR) is crucial to life by regulating the cellular response to the stress in the endoplasmic reticulum (ER) imposed by abiotic and biotic cues such as heat shock and viral infection. The inositol requiring enzyme 1 (IRE1) signaling pathway activated by the IRE1-mediated unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans, is the most ancient branch of the UPR. In this study, we systematically examined yeast IRE1p-HAC1, plant IRE1A/IRE1B-bZIP60 and human hIRE1-XBP1 pairs. We found that, unlike bZIP60, XBP1 is unable to functionally swap HAC1p in yeast, and that the inter-species heterotypic interactions among HAC1p, bZIP60 and XBP1 are not permitted. These data demonstrate evolutionary divergence of the downstream signaling of IRE1-bZIP. We also discovered that the dual cytosolic domains of plant IRE1s act in vivo in a mechanism consistent with IRE1p and hIRE1, and that plant IRE1B not only interacts with IRE1p but also forms typical IRE1 dynamic foci in yeast. Thus, the upstream components of the IRE1 signaling branch including IRE1 activation and action mechanisms are highly conserved. Taken together these data advance the molecular understanding of evolutionary divergence and conservation of the IRE1 signaling pathway across kingdoms. PMID:27256815

  13. IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses

    PubMed Central

    Blanco, Francisca; Boatwright, Jon Lucas; Moreno, Ignacio; Jordan, Melissa R.; Chen, Yani; Brandizzi, Federica; Dong, Xinnian

    2012-01-01

    Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in

  14. Cadherin 2/4 signaling via PTP1B and catenins is crucial for nucleokinesis during radial neuronal migration in the neocortex

    PubMed Central

    Martinez-Garay, Isabel; Gil-Sanz, Cristina; Franco, Santos J.; Espinosa, Ana; Molnár, Zoltán

    2016-01-01

    Cadherins are crucial for the radial migration of excitatory projection neurons into the developing neocortical wall. However, the specific cadherins and the signaling pathways that regulate radial migration are not well understood. Here, we show that cadherin 2 (CDH2) and CDH4 cooperate to regulate radial migration in mouse brain via the protein tyrosine phosphatase 1B (PTP1B) and α- and β-catenins. Surprisingly, perturbation of cadherin-mediated signaling does not affect the formation and extension of leading processes of migrating neocortical neurons. Instead, movement of the cell body and nucleus (nucleokinesis) is disrupted. This defect is partially rescued by overexpression of LIS1, a microtubule-associated protein that has previously been shown to regulate nucleokinesis. Taken together, our findings indicate that cadherin-mediated signaling to the cytoskeleton is crucial for nucleokinesis of neocortical projection neurons during their radial migration. PMID:27151949

  15. The power-density spectra of some nB(n + 1) B block coded signals

    NASA Astrophysics Data System (ADS)

    Morgenstern, G.

    1986-08-01

    In many cases, there arises the problem of adapting the power-density spectrum of a binary signal to a transmission channel or of adding redundancy. In such cases, a utilization of the considered block codes can be useful. For these applications, n binary symbols are combined in a block and converted with the aid of a code table. The current paper presents a summary of a technical report provided by Morgenstern (1985), who considers the code relations for 1B2B, 3B4B, and 5B6B block codes. The spectral power densities of signals coded according to these relations are derived, taking into account an arbitrary pulse shape and rectangular pulses. It is pointed out that the 5B6B block code is employed in connection with optical fiber transmissions.

  16. Modulation of Cellular Insulin signaling and PTP1B effects by lipid metabolites in skeletal muscle cells

    PubMed Central

    Obanda, Diana N.; Cefalu, William T.

    2015-01-01

    Normal glucose regulation is achieved by having adequate insulin secretion and effective glucose uptake/disposal. Excess lipids in peripheral tissues: skeletal muscle, liver and adipose tissue may attenuate insulin signaling through the protein kinase B (AKt/PKB) pathway and upregulate protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signaling. We studied accumulation of lipid metabolites [triglycerides (TAGs), diglycerides (DAGs)] and ceramides in relation to insulin signaling and expression and phosphorylation of PTP1B by preincubating rat skeletal muscle cells (L6 myotubes) with 3 saturated and 3 unsaturated free fatty acids (FFAs) (200uM). Cells were also evaluated in the presence of wortmannin an inhibitor of Phosphatidylinositol 3-kinases and thus AKt (0–100nM). Unsaturated FFAs increased DAGs, TAGs and PTP1B expression significantly but, cells remained insulin sensitive as assessed by robust AKt and PTP1B phosphorylation at Serine (Ser) 50, Ser 398 and Tyrosine (Tyr) 152. Saturated palmitic and stearic acids increased ceramides, upregulated PTP1B and had AKt and PTP1B phosphorylation at Ser 50 impaired. With increasing palmitic acid dose (0–200 uM), we show a significant positive correlation between phosphorylation levels of AKt and of PTP1B at Ser 50 (R2=0.84; P<0.05). The same was observed with increasing wortmannin dose (R2=0.73; P<0.05). Only FFAs that increased ceramides caused impairment of AKt and PTP1B phosphorylation at Ser 50. PTP1B overexpression in presence of excess lipids may not directly cause insulin resistance unless it is accompanied by decreased PTP1B phosphorylation. A clear relationship between PTP1B phosphorylation levels at Ser 50 and its negative effect on insulin signaling is shown. PMID:23481236

  17. Overexpression of JARID1B promotes differentiation via SHIP1/AKT signaling in human hypopharyngeal squamous cell carcinoma.

    PubMed

    Zhang, Jisheng; An, Xiaofei; Han, Yafei; Ma, Rui; Yang, Kun; Zhang, Lu; Chi, Jingwei; Li, Wei; Llobet-Navas, David; Xu, Yan; Jiang, Yan

    2016-01-01

    Histone H3 (H3K4) demethylase JARID1B is aberrantly upregulated in many types of tumor and has been proposed to function as oncogene. Here we show that JARID1B is elevated in moderate and high-differentiated human hypopharyngeal squamous cell carcinoma (HPSCC) compared with low-differentiated HPSCC. Overexpression of JARID1B in FaDu cells increased epithelial differentiation marker K10 expression and inhibited cell proliferation. JARID1B and K10 mRNA expression is high correlated in HPSCC patients. Mechanistically, we found JARID1B directly bound to PI3K/AKT signaling inhibitor SHIP1 gene promoter and decreased SHIP1 gene expression. Activation of downstream AKT resulted in increased β-catenin signaling, by which promoted target genes Fra-1 and Jun, together with other AP-1 transcription factors, leading to K10 expression. Forced expression of SHIP1 rescued JARID1B-induced phenotypes on FaDu cell differentiation and proliferation. Taken together, our findings provide first evidence that elevated expression of JARID1B has a critical role in promoting HPSCC differentiation and inhibiting proliferation, suggesting JARID1B may function as a tumor suppressor in squamous cell cancers and implying a novel important therapeutic strategy of HPSCC. PMID:27584795

  18. Overexpression of JARID1B promotes differentiation via SHIP1/AKT signaling in human hypopharyngeal squamous cell carcinoma

    PubMed Central

    Zhang, Jisheng; An, Xiaofei; Han, Yafei; Ma, Rui; Yang, Kun; Zhang, Lu; Chi, Jingwei; Li, Wei; Llobet-Navas, David; Xu, Yan; Jiang, Yan

    2016-01-01

    Histone H3 (H3K4) demethylase JARID1B is aberrantly upregulated in many types of tumor and has been proposed to function as oncogene. Here we show that JARID1B is elevated in moderate and high-differentiated human hypopharyngeal squamous cell carcinoma (HPSCC) compared with low-differentiated HPSCC. Overexpression of JARID1B in FaDu cells increased epithelial differentiation marker K10 expression and inhibited cell proliferation. JARID1B and K10 mRNA expression is high correlated in HPSCC patients. Mechanistically, we found JARID1B directly bound to PI3K/AKT signaling inhibitor SHIP1 gene promoter and decreased SHIP1 gene expression. Activation of downstream AKT resulted in increased β-catenin signaling, by which promoted target genes Fra-1 and Jun, together with other AP-1 transcription factors, leading to K10 expression. Forced expression of SHIP1 rescued JARID1B-induced phenotypes on FaDu cell differentiation and proliferation. Taken together, our findings provide first evidence that elevated expression of JARID1B has a critical role in promoting HPSCC differentiation and inhibiting proliferation, suggesting JARID1B may function as a tumor suppressor in squamous cell cancers and implying a novel important therapeutic strategy of HPSCC. PMID:27584795

  19. The Adenovirus Type 5 E1B-55K Oncoprotein Actively Shuttles in Virus-Infected Cells, Whereas Transport of E4orf6 Is Mediated by a CRM1-Independent Mechanism

    PubMed Central

    Dosch, Tanja; Horn, Florian; Schneider, Grit; Krätzer, Friedrich; Dobner, Thomas; Hauber, Joachim; Stauber, Roland H.

    2001-01-01

    The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the “driving force” for adenoviral late mRNA export. PMID:11356976

  20. BMPR1a and BMPR1b Signaling Exert Opposing Effects on Gliosis after Spinal Cord Injury

    PubMed Central

    Sahni, Vibhu; Mukhopadhyay, Abhishek; Tysseling, Vicki; Hebert, Amy; Birch, Derin; Mcguire, Tammy L.; Stupp, Samuel I.; Kessler, John A.

    2011-01-01

    Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that is beneficial and a subsequent formation of a dense scar. We investigated the role of bone morphogenetic protein (BMP) signaling in gliosis after SCI and find that BMPR1a and BMPR1b signaling exerts opposing effects on hypertrophy. Conditional ablation of BMPR1a from glial fibrillary acidic protein (GFAP)-expressing cells leads to defective astrocytic hypertrophy, increased infiltration by inflammatory cells, and reduced axon density. BMPR1b-null mice conversely develop “hyperactive” reactive astrocytes and consequently have smaller lesion volumes. The effects of ablation of either receptor are reversed in the double knock-out animals. These findings indicate that BMPR1a and BMPR1b exert directly opposing effects on the initial reactive astrocytic hypertrophy. Also, BMPR1b knock-out mice have an attenuated glial scar in the chronic stages following injury, suggesting that it has a greater role in glial scar progression. To elucidate the differing roles of the two receptors in astrocytes, we examined the effects of ablation of either receptor in serum-derived astrocytes in vitro. We find that the two receptors exert opposing effects on the posttranscriptional regulation of astrocytic microRNA-21. Further, overexpression of microRNA-21 in wild-type serum-derived astrocytes causes a dramatic reduction in cell size accompanied by reduction in GFAP levels. Hence, regulation of microRNA-21 by BMP signaling provides a novel mechanism for regulation of astrocytic size. Targeting specific BMPR subunits for therapeutic purposes may thus provide an approach for manipulating gliosis and enhancing functional outcomes after SCI. PMID:20130193

  1. Isolation of chromatin from dysfunctional telomeres reveals an important role for Ring1b in NHEJ-mediated chromosome fusions.

    PubMed

    Bartocci, Cristina; Diedrich, Jolene K; Ouzounov, Iliana; Li, Julia; Piunti, Andrea; Pasini, Diego; Yates, John R; Lazzerini Denchi, Eros

    2014-05-22

    When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh) in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ)-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation. PMID:24813883

  2. Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

    PubMed

    Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

    2014-12-01

    Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis.

  3. Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

    PubMed

    Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

    2014-12-01

    Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis. PMID:25193465

  4. Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT1B receptors in C6-glial cells: similarities with the human 5-HT1B receptor

    PubMed Central

    Pauwels, Petrus J; Wurch, Thierry; Palmier, Christiane; Colpaert, Francis C

    1998-01-01

    ]-GTPγS binding at concentrations relevant to their binding affinity for the gp 5-HT1B receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5-HT1B receptors; pA2 values were 9.74 and 8.73, respectively when 5-HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan-mediated inhibition of forskolin-stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pKB: 5.87) at gp 5-HT1B receptors.In conclusion, the recombinant gp 5-HT1B receptor shares important pharmacological similarities with the recombinant h 5-HT1B receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists. PMID:9484854

  5. Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT1B receptors in C6-glial cells: similarities with the human 5-HT1B receptor.

    PubMed

    Pauwels, P J; Wurch, T; Palmier, C; Colpaert, F C

    1998-01-01

    relevant to their binding affinity for the gp 5-HT1B receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5-HT1B receptors; pA2 values were 9.74 and 8.73, respectively when 5-HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan-mediated inhibition of forskolin-stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pK(B): 5.87) at gp 5-HT1B receptors. 5. In conclusion, the recombinant gp 5-HT1B receptor shares important pharmacological similarities with the recombinant h 5-HT1B receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists. PMID:9484854

  6. Tumor-related alternatively spliced Rac1b is not regulated by Rho-GDP dissociation inhibitors and exhibits selective downstream signaling.

    PubMed

    Matos, Paulo; Collard, John G; Jordan, Peter

    2003-12-12

    Rac1 is a member of the Rho family of small GTPases, which control signaling pathways that regulate actin cytoskeletal dynamics and gene transcription. Rac1 is activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. In addition, Rho-GDP dissociation inhibitors (Rho-GDIs) can inhibit Rac1 by sequestering it in the cytoplasm. We have found previously that colorectal tumors express an alternatively spliced variant, Rac1b, containing 19 additional amino acids following the switch II region. Here we characterized the regulation and downstream signaling of Rac1b. Although little Rac1b protein is expressed in cells, the amount of activated Rac1b protein often exceeds that of activated Rac1, suggesting that Rac1b contributes significantly to the downstream signaling of Rac in cells. The regulation of both Rac1 and Rac1b activities is dependent on guanine nucleotide exchange factors and GTPase-activating proteins, but the difference in their activation is mainly determined by the inability of Rac1b to interact with Rho-GDI. As a consequence, most Rac1b remains bound to the plasma membrane and is not sequestered by Rho-GDI in the cytoplasm. Unlike Rac1, activated Rac1b is unable to induce lamellipodia formation and is unable to bind and activate p21-activated protein kinase nor activate the downstream protein kinase JNK. However, both Rac1 and Rac1b are able to activate NFkappaB to the same extent. These data suggest that alternative splicing of Rac1 leads to a highly active Rac variant that differs in regulation and downstream signaling.

  7. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    PubMed

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE.

  8. Evidence for hSNM1B/Apollo functioning in the HSP70 mediated DNA damage response.

    PubMed

    Anders, Marco; Mattow, Jens; Digweed, Martin; Demuth, Ilja

    2009-06-01

    The hSNM1B/Apollo protein is involved in the cellular response to DNA-damage as well as in the maintenance of telomeres during S-phase. TRF2 has been shown to interact physically with hSNM1B. As a core component of shelterin, TRF2 functions in organization and protection of telomeres. However, TRF2 was also shown to have a role in the early DNA-damage response, suggesting that hSNM1B and TRF2 cooperate in this dual function. Here we have used Tandem-Affinity-Purification in combination with mass spectrometry to identify additional binding partners of hSNM1B. This revealed HSC70, HSP72, HSP60 and beta-Tubulin to be hSNM1B-interactors. We have confirmed the interaction of hSNM1B and HSP70 in co-immunoprecipitation assays and found that hSNM1B binds to a C-terminal fragment of HSP72, known to contain the substrate binding domain. Depletion of HSP72 in human fibroblasts resulted in a significant reduction of nuclear hSNM1B foci. We also found the phosphorylation of CHK1 at serine 317 to be attenuated in response to UVC irradiation as a consequence of hSNM1B depletion, a result which extends our previous findings on the DNA-damage response function of hSNM1B. HSP70 chaperones have been implicated in the maintenance of genome stability and their expression is often aberrant in cancer. Our results presented here, suggest that the role in genome stability might not be specific to HSP70 but rather can be attributed, at least in part, to hSNM1B. This, together with its stimulating effect on ATM and ATR substrate phosphorylation in response to DNA-damage qualify hSNM1B as a putative target in cancer therapy.

  9. Aldo-keto reductase 1b7, a novel marker for renin cells, is regulated by cyclic AMP signaling

    PubMed Central

    Lin, Eugene E.; Pentz, Ellen S.; Sequeira-Lopez, Maria Luisa S.

    2015-01-01

    We previously identified aldo-keto reductase 1b7 (AKR1B7) as a marker for juxtaglomerular renin cells in the adult mouse kidney. However, the distribution of renin cells varies dynamically, and it was unknown whether AKR1B7 maintains coexpression with renin in response to different developmental, physiological, and pathological situations, and furthermore, whether similar factor(s) simultaneously regulate both proteins. We show here that throughout kidney development, AKR1B7 expression—together with renin—is progressively restricted in the kidney arteries toward the glomerulus. Subsequently, when formerly renin-expressing cells reacquire renin expression, AKR1B7 is reexpressed as well. This pattern of coexpression persists in extreme pathological situations, such as deletion of the genes for aldosterone synthase or Dicer. However, the two proteins do not colocalize within the same organelles: renin is found in the secretory granules, whereas AKR1B7 localizes to the endoplasmic reticulum. Interestingly, upon deletion of the renin gene, AKR1B7 expression is maintained in a pattern mimicking the embryonic expression of renin, while ablation of renin cells resulted in complete abolition of AKR1B7 expression. Finally, we demonstrate that AKR1B7 transcription is controlled by cAMP. Cultured cells of the renin lineage reacquire the ability to express both renin and AKR1B7 upon elevation of intracellular cAMP. In vivo, deleting elements of the cAMP-response pathway (CBP/P300) results in a stark decrease in AKR1B7- and renin-positive cells. In summary, AKR1B7 is expressed within the renin cell throughout development and perturbations to homeostasis, and AKR1B7 is regulated by cAMP levels within the renin cell. PMID:26180185

  10. ROS and ROS-Mediated Cellular Signaling

    PubMed Central

    Zhang, Jixiang; Wang, Xiaoli; Vikash, Vikash; Ye, Qing; Wu, Dandan; Liu, Yulan; Dong, Weiguo

    2016-01-01

    It has long been recognized that an increase of reactive oxygen species (ROS) can modify the cell-signaling proteins and have functional consequences, which successively mediate pathological processes such as atherosclerosis, diabetes, unchecked growth, neurodegeneration, inflammation, and aging. While numerous articles have demonstrated the impacts of ROS on various signaling pathways and clarify the mechanism of action of cell-signaling proteins, their influence on the level of intracellular ROS, and their complex interactions among multiple ROS associated signaling pathways, the systemic summary is necessary. In this review paper, we particularly focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins (NF-κB, MAPKs, Keap1-Nrf2-ARE, and PI3K-Akt), ion channels and transporters (Ca2+ and mPTP), and modifying protein kinase and Ubiquitination/Proteasome System. PMID:26998193

  11. Repulsive axon guidance by Draxin is mediated by protein Kinase B (Akt), glycogen synthase kinase-3β (GSK-3β) and microtubule-associated protein 1B.

    PubMed

    Meli, Rajeshwari; Weisová, Petronela; Propst, Friedrich

    2015-01-01

    Draxin is an important axon guidance cue necessary for the formation of forebrain commissures including the corpus callosum, but the molecular details of draxin signaling are unknown. To unravel how draxin signals are propagated we used murine cortical neurons and genetic and pharmacological approaches. We found that draxin-induced growth cone collapse critically depends on draxin receptors (deleted in colorectal cancer, DCC), inhibition of protein kinase B/Akt, activation of GSK-3β (glycogen synthase kinase-3β) and the presence of microtubule-associated protein MAP1B. This study, for the first time elucidates molecular events in draxin repulsion, links draxin and DCC to MAP1B and identifies a novel MAP1B-depenent GSK-3β pathway essential for chemo-repulsive axon guidance cue signaling.

  12. Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association

    PubMed Central

    Park, Pil Whan; Ahn, Jeong Yeal

    2016-01-01

    Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B. PMID:27382360

  13. The Clinical Relevance of the miR-197/CKS1B/STAT3-mediated PD-L1 Network in Chemoresistant Non-small-cell Lung Cancer

    PubMed Central

    Fujita, Yu; Yagishita, Shigehiro; Hagiwara, Keitaro; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Tsuta, Koji; Nokihara, Hiroshi; Tamura, Tomohide; Asamura, Hisao; Kawaishi, Makoto; Kuwano, Kazuyoshi; Ochiya, Takahiro

    2015-01-01

    Programmed cell death ligand-1 (PD-L1) has recently gained considerable attention for its role in tumor immune escape. Here, we identify a miR-197/CKS1B/STAT3-mediated PD-L1 network in chemoresistant non-small-cell lung cancer (NSCLC), independent of immunoinhibitory signals. miR-197 is downregulated in platinum-resistant NSCLC specimens, resulting in the promotion of chemoresistance, tumorigenicity, and pulmonary metastasis in vitro and in vivo. Mechanistic investigations reveal that a miR-197-mediated CKS1B/STAT3 axis exerts tumor progression regulated by various oncogenic genes (Bcl-2, c-Myc, and cyclin D1), and PD-L1 is a putative biomarker of this axis. Furthermore, we demonstrate that a miR-197 mimic sensitizes PD-L1high drug-resistant cells to chemotherapy. These results indicate that the biological interaction between PD-L1 and chemoresistance occurs through the microRNA regulatory cascade. More importantly, expression levels of miR-197 are inversely correlated with PD-L1 expression (n = 177; P = 0.026) and are associated with worse overall survival (P = 0.015). Our discoveries suggest that the miR-197/CKS1B/STAT3-mediated network can drive tumor PD-L1 expression as a biomarker of this cascade, and miR-197 replacement therapy may be a potential treatment strategy for chemoresistant NSCLC. PMID:25597412

  14. Pronephric tubule morphogenesis in zebrafish depends on Mnx mediated repression of irx1b within the intermediate mesoderm.

    PubMed

    Ott, Elisabeth; Wendik, Björn; Srivastava, Monika; Pacho, Frederic; Töchterle, Sonja; Salvenmoser, Willi; Meyer, Dirk

    2016-03-01

    Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros.

  15. Transposon-mediated alteration of TaMATE1B expression in wheat confers constitutive citrate efflux from root apices.

    PubMed

    Tovkach, Andriy; Ryan, Peter R; Richardson, Alan E; Lewis, David C; Rathjen, Tina M; Ramesh, Sunita; Tyerman, Stephen D; Delhaize, Emmanuel

    2013-02-01

    The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3' regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance.

  16. The pituitary mediates the anxiolytic-like effects of the vasopressin V1B receptor antagonist, SSR149415, in a social interaction test in rats.

    PubMed

    Shimazaki, Toshiharu; Iijima, Michihiko; Chaki, Shigeyuki

    2006-08-14

    A vasopressin V(1B) receptor antagonist has been shown to exhibit anxiolytic effects in a variety of animal models of anxiety. In the present study, we examined the involvement of the pituitary in the anxiolytic effects of a vasopressin V(1B) receptor antagonist by conducting a social interaction test in rats. In the sham-operated rats, both the vasopressin V(1B) receptor antagonist SSR149415 and the benzodiazepine chlordiazepoxide significantly increased the social behavior of a pair of unfamiliar rats, and the blood adrenocorticotropic hormone levels were markedly increased during the social interaction test. Hypophysectomy also increased the length of time that the animals engaged in social behavior to the same extent as that observed after treatment of the sham-operated rats with anxiolytics. However, while chlordiazepoxide further increased the duration of social interaction in the hypophysectomized rats, the anxiolytic effects of SSR149415 was no longer observed in these animals. These results suggest that the anxiolytic effects of the vasopressin V(1B) receptor antagonist in the social interaction test are mediated through blockade of the vasopressin V(1B) receptor in the pituitary.

  17. 3.5 keV X-ray line signal from dark matter decay in local U(1) B- L extension of Zee-Babu model

    NASA Astrophysics Data System (ADS)

    Baek, Seungwon

    2015-08-01

    We consider a local U(1) B- L extension of Zee-Babu model to explain the recently observed 3.5 keV X-ray line signal. The model has three Standard model (SM)-singlet Dirac fermions with different U(1) B- L charges. A complex scalar field charged under U(1) B- L is introduced to break the U(1) B- L symmetry. After U(1) B- L symmetry breaking a remnant discrete symmetry stabilizes the lightest state of the Dirac fermions, which can be a stable dark matter (DM). The second lightest state, if mass splitting with the stable DM is about 3.5 keV, decays dominantly to the stable DM and 3.5 keV photon through two-loop diagrams, explaining the X-ray line signal. Two-loop suppression of the decay amplitude makes its lifetime much longer than the age of the universe and it can be a decaying DM candidate in large parameter region. We also introduce a real scalar field which is singlet under both the SM and U(1) B- L and can explain the current relic abundance of the Dirac fermionic DMs. If the mixing with the SM Higgs boson is small, it does not contribute to DM direct detection. The main contribution to the scattering of DM off atomic nuclei comes from the exchange of U(1) B- L gauge boson, Z ', and is suppressed below current experimental bound when Z' mass is heavy (≳10 TeV). If the singlet scalar mass is about 0.1-10 MeV, DM self-interaction can be large enough to solve small scale structure problems in simulations with the cold DM, such as, the core-vs-cusp problem and too-big-to-fail problem.

  18. Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17β-glucuronide transport

    PubMed Central

    Gui, Chunshan; Wahlgren, Brett; Lushington, Gerald H.; Hagenbuch, Bruno

    2009-01-01

    Evidence shows that drug-drug interactions can occur at the level of drug transporters such as the organic anion transporting polypeptides (OATPs), a group of membrane solute carriers that mediate the sodium-independent transport of a wide range of amphipathic organic compounds. The polyspecific OATP1B1 is exclusively expressed at the basolateral membrane of hepatocytes and mediates uptake of amphipathic organic compounds from blood into hepatocytes. Nuclear receptors are ligand-activated transcription factors that play an important role in xenobiotic disposition and human diseases. Quite a few nuclear receptor ligands interact with transport proteins. A high-resolution three-dimensional structure is critical to understand the polyspecificity of OATP1B1 to predict and prevent adverse drug-drug interactions. Unfortunately there are no crystal structures of OATPs/Oatps available to date. Therefore, in this study we attempted to elucidate the characteristics of the substrate binding site of OATP1B1 based on small molecules interacting with it. First, we identified inhibitors of the OATP1B1 model substrate estradiol-17β-glucuronide from about forty nuclear receptor ligands. Among them, GW1929, paclitaxel and troglitazone were strong inhibitors, while 5α-androstane, 5α-androstane-3β, 17β-diol-17-hexahydrobenzoate and estradiol-3-benzoate were weak inhibitors. Then, we selected 25 compounds and performed inhibition kinetic studies to identify competitive inhibitors and determine their Ki values which ranged from submicromolar to submillimolar. Finally, we performed CoMFA analysis on the identified competitive inhibitors. The CoMFA results indicate that the substrate binding site of OATP1B1 consists of a large hydrophobic middle part with basic residues at both ends that could be very important for substrate binding. PMID:19427586

  19. Application of a fuzzy neural network model in predicting polycyclic aromatic hydrocarbon-mediated perturbations of the Cyp1b1 transcriptional regulatory network in mouse skin

    SciTech Connect

    Larkin, Andrew; Siddens, Lisbeth K.; Krueger, Sharon K.; Tilton, Susan C.; Waters, Katrina M.; Williams, David E.; Baird, William M.

    2013-03-01

    Polycyclic aromatic hydrocarbons (PAHs) are present in the environment as complex mixtures with components that have diverse carcinogenic potencies and mostly unknown interactive effects. Non-additive PAH interactions have been observed in regulation of cytochrome P450 (CYP) gene expression in the CYP1 family. To better understand and predict biological effects of complex mixtures, such as environmental PAHs, an 11 gene input-1 gene output fuzzy neural network (FNN) was developed for predicting PAH-mediated perturbations of dermal Cyp1b1 transcription in mice. Input values were generalized using fuzzy logic into low, medium, and high fuzzy subsets, and sorted using k-means clustering to create Mamdani logic functions for predicting Cyp1b1 mRNA expression. Model testing was performed with data from microarray analysis of skin samples from FVB/N mice treated with toluene (vehicle control), dibenzo[def,p]chrysene (DBC), benzo[a]pyrene (BaP), or 1 of 3 combinations of diesel particulate extract (DPE), coal tar extract (CTE) and cigarette smoke condensate (CSC) using leave-one-out cross-validation. Predictions were within 1 log{sub 2} fold change unit of microarray data, with the exception of the DBC treatment group, where the unexpected down-regulation of Cyp1b1 expression was predicted but did not reach statistical significance on the microarrays. Adding CTE to DPE was predicted to increase Cyp1b1 expression, whereas adding CSC to CTE and DPE was predicted to have no effect, in agreement with microarray results. The aryl hydrocarbon receptor repressor (Ahrr) was determined to be the most significant input variable for model predictions using back-propagation and normalization of FNN weights. - Highlights: ► Tested a model to predict PAH mixture-mediated changes in Cyp1b1 expression ► Quantitative predictions in agreement with microarrays for Cyp1b1 induction ► Unexpected difference in expression between DBC and other treatments predicted ► Model predictions

  20. Primary cilia signaling mediates intraocular pressure sensation.

    PubMed

    Luo, Na; Conwell, Michael D; Chen, Xingjuan; Kettenhofen, Christine Insinna; Westlake, Christopher J; Cantor, Louis B; Wells, Clark D; Weinreb, Robert N; Corson, Timothy W; Spandau, Dan F; Joos, Karen M; Iomini, Carlo; Obukhov, Alexander G; Sun, Yang

    2014-09-01

    Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-β, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.

  1. Photoreceptor phagocytosis is mediated by phosphoinositide signaling.

    PubMed

    Mustafi, Debarshi; Kevany, Brian M; Genoud, Christel; Bai, Xiaodong; Palczewski, Krzysztof

    2013-11-01

    Circadian oscillations in peripheral tissues, such as the retinal compartment of the eye, are critical to anticipating changing metabolic demands. Circadian shedding of retinal photoreceptor cell discs with subsequent phagocytosis by the neighboring retinal pigmented epithelium (RPE) is essential for removal of toxic metabolites and lifelong survival of these postmitotic neurons. Defects in photoreceptor phagocytosis can lead to severe retinal pathology, but the biochemical mechanisms remain poorly defined. By first documenting a 2.8-fold burst of photoreceptor phagocytosis events in the mouse eye in the morning compared with the afternoon by serial block face imaging, we established time points to assess transcriptional readouts by RNA sequencing (RNA-Seq). We identified 365 oscillating protein-coding transcripts that implicated the phosphoinositide lipid signaling network mediating the discrete steps of photoreceptor phagocytosis. Moreover, examination of overlapping cistromic sites by core clock transcription factors and promoter elements of these effector genes provided a functional basis for the circadian cycling of these transcripts. RNA-Seq also revealed oscillating expression of 16 long intergenic noncoding RNAs and key histone modifying enzymes critical for circadian gene expression. Our phenotypic and genotypic characterization reveals a complex global landscape of overlapping and temporally controlled networks driving the essential circadian process in the eye.

  2. The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling

    PubMed Central

    Drusenheimer, Nadja; Migdal, Bernhard; Jäckel, Sandra; Tveriakhina, Lena; Scheider, Kristina; Schulz, Katharina; Gröper, Jieny; Köhrer, Karl; Klein, Thomas

    2015-01-01

    CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT

  3. Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence.

    PubMed

    Lee, Sang-Hwa; Sakuraba, Yasuhito; Lee, Taeyoung; Kim, Kyu-Won; An, Gynheung; Lee, Han Yong; Paek, Nam-Chon

    2015-06-01

    Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA-responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b-1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence-promoting conditions. oscoi1b-1 mutants stayed green during dark-induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence-associated genes were downregulated in oscoi1b-1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1-1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b-1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1-mediated JA signaling affects spikelet fertility and grain filling.

  4. ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

    PubMed Central

    Su, Gloria H.; Bansal, Ravi; Murphy, Kathleen M.; Montgomery, Elizabeth; Yeo, Charles J.; Hruban, Ralph H.; Kern, Scott E.

    2001-01-01

    DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene. PMID:11248065

  5. Shiga toxin (Stx)1B and Stx2B induce von Willebrand factor secretion from human umbilical vein endothelial cells through different signaling pathways.

    PubMed

    Liu, Fang; Huang, Jing; Sadler, J Evan

    2011-09-22

    Diarrhea-associated hemolytic uremic syndrome (D(+)HUS) is caused by the ingestion of Escherichia coli that produce Shiga toxin (Stx), which is composed of a cytotoxic A subunit and pentameric B subunits that bind globotriaosylceramide on susceptible cells. Stx occurs in 2 types, Stx1 and Stx2. B subunits of either type stimulate von Willebrand factor (VWF) secretion from human umbilical vein endothelial cells (HUVECs), and Stx2B can cause thrombotic microangiopathy in Adamts13(-/-) mice. We have now determined that Stx1B and Stx2B activate different signaling pathways in HUVECs. VWF secretion induced by Stx1B is associated with a transient rise in intracellular Ca(2+) level that is blocked by chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, removal of extracellular Ca(2+), the phospholipase C inhibitor U73122, the protein kinase inhibitor staurosporine, or small interfering RNA knockdown of protein kinase Cα. In contrast, Stx2B-induced VWF secretion is associated with activation of protein kinase A (PKA) and is blocked by the PKA inhibitor H89 or small interfering RNA knockdown of PKA. Stx2B does not increase cAMP levels and may activate PKA by a cAMP-independent mechanism. The activation of distinct signaling pathways may be relevant to understanding why E coli that express Stx2 are more likely to cause D(+)HUS than are E coli expressing only Stx1.

  6. Requirement of TGFβ Signaling for SMO-mediated Carcinogenesis*

    PubMed Central

    Fan, Qipeng; He, Miao; Sheng, Tao; Zhang, Xiaoli; Sinha, Mala; Luxon, Bruce; Zhao, Xingbo; Xie, Jingwu

    2010-01-01

    Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. At present, the molecular mechanism of Hh signaling-mediated carcinogenesis is not completely understood. Using a mouse model (K14cre/R26SmoM2) of SMO-mediated basal cell carcinoma development, we identified TGFβ2 as a major Hh-regulated gene. TGFβ2 expression was high in the keratinocytes, with activated TGFβ signaling (indicated by elevated expression of phosphorylated SMAD2/3) detected in both tumor and stroma. The significance of TGFβ signaling for SMO function was demonstrated in two assays. Down-regulation of TGFβ2 expression prevented Hh signaling-dependent osteoblast differentiation and motor neuron differentiation. Furthermore, inhibition of TGFβ signaling by TGFβ receptor I inhibitor SD208 significantly reduced tumor area in K14cre/R26SmoM2 mice. Tumor shrinkage in mice was associated with an increased number of lymphocytes, suggesting an immune suppression role of TGFβ signaling. The relevance of our results to human cancer is reflected by the fact that human basal cell carcinomas, which almost always harbor activated Hh signaling, have activated TGFβ signaling, as indicated by high levels of phosphorylated SMAD2 and SMAD3 in tumor and stroma. Together, our data indicate that TGFβ signaling is critical for Hh signaling-mediated carcinogenesis. PMID:20858897

  7. ERK5 Mediated Signalling in Diabetic Retinopathy

    PubMed Central

    Wu, Yuexiu; Chakrabarti, Subrata

    2015-01-01

    Diabetic retinopathy is the lead among causes of blindness in North America. Glucose-induced endothelial injury is the most important cause of diabetic retinopathy and other vascular complications. Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1 (BMK1), is a member of mitogen-activated protein kinases (MAPK) family. Physiologically, it is critical for cardiovascular development and maintenance of the endothelial cell integrity. Extracellular signal-regulated kinase 5 is protective for endothelial cells under stimulation and stress. Decreased activation of ERK5 results in increased endothelial cell death. Extracellular signal-regulated kinase 5 signaling may be subject to alteration by hyperglycemia, while signaling pathway including ERK5 may be subject to alteration during pathogenesis of diabetic complications. In this review, the role of ERK5 in diabetic macro- and microvascular complications with a focus on diabetic retinopathy are summarized and discussed. PMID:25861671

  8. [Zinc signaling-mediated regulation of dentin and periodontal tissues].

    PubMed

    Fukada, Toshiyuki; Idaira, Yayoi; Shimoda, Shinji; Asada, Yoshinobu

    2015-12-01

    An essential trace element zinc is required for variety of cellular functions and physiological responses, therefore, downregulation of zinc homeostasis cause serious problems in health, such as growth retardation and abnormal bone formation. Recent technical advances contributed to reveal that zinc ion regulated by zinc transporters acts as a signaling mediator, called zinc signaling that involves in mammalian physiology and pathogenesis. This review will address the current understanding of the roles of zinc signaling in regulation of dentin formation and periodontal tissues homeostasis.

  9. Dark matter signals in deflected mirage mediation

    SciTech Connect

    Holmes, Michael

    2010-02-10

    We investigate the parameter space of a specific class of model within the deflected mirage mediation (DMM) scenario. We look at neutralino properties and compute the thermal relic density as well as interaction rates with xenon direct detection experiments. We find that there are portions of the parameter space which are in line with the current WMAP constraints. Further we find that none of the investigated parameter space is in conflict with current bounds from the Xenon10 experiment and that future large-scale liquid xenon experiments will probe a large portion of the model space.

  10. LHC Signals of Pure Gravity Mediation

    NASA Astrophysics Data System (ADS)

    Feldstein, Brian

    2013-05-01

    Evidence is mounting that natural supersymmetry at the weak scale is not realized in nature. This evidence comes from collider searches, a lack of new flavor changing neutral current effects, and now also the size of the measured Higgs mass. On the other hand, string theory suggests that supersymmetry might be present at some energy scale, and gauge coupling unification and dark matter imply that that energy scale may be relatively low. The simplest model to address all of these hints is arguably "pure gravity mediation", in which the scalar superpartner masses are taken to be perhaps 100 TeV, with gauginos automatically acquiring loop factor suppressed masses of order TeV. The gauginos might then be the only superpartners accessible to the LHC. Unification and LSP dark matter are maintained (with a wino LSP) at the cost of a 10-5 or 10-6 fine tuning. Here I will discuss the structure and LHC phenomenology of pure gravity mediation.

  11. 6β-Hydroxytestosterone, a Cytochrome P450 1B1-Testosterone-Metabolite, Mediates Angiotensin II-Induced Renal Dysfunction in Male Mice.

    PubMed

    Pingili, Ajeeth K; Thirunavukkarasu, Shyamala; Kara, Mehmet; Brand, David D; Katsurada, Akemi; Majid, Dewan S A; Navar, L Gabriel; Gonzalez, Frank J; Malik, Kafait U

    2016-05-01

    6β-Hydroxytestosterone, a cytochrome P450 1B1-derived metabolite of testosterone, contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the maintenance of renal homeostasis, development of hypertension, and end-organ damage, this study was conducted to determine the contribution of 6β-hydroxytestosterone to angiotensin II actions on water consumption and renal function in male Cyp1b1(+/+) and Cyp1b1(-/-) mice. Castration of Cyp1b1(+/+) mice or Cyp1b1(-/-) gene disruption minimized the angiotensin II-induced increase in water consumption, urine output, proteinuria, and sodium excretion and decreases in urine osmolality. 6β-Hydroxytestosterone did not alter angiotensin II-induced increases in water intake, urine output, proteinuria, and sodium excretion or decreases in osmolality in Cyp1b1(+/+) mice, but restored these effects of angiotensin II in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice. Cyp1b1 gene disruption or castration prevented angiotensin II-induced renal fibrosis, oxidative stress, inflammation, urinary excretion of angiotensinogen, expression of angiotensin II type 1 receptor, and angiotensin-converting enzyme. 6β-Hydroxytestosterone did not alter angiotensin II-induced renal fibrosis, inflammation, oxidative stress, urinary excretion of angiotensinogen, expression of angiotensin II type 1 receptor, or angiotensin-converting enzyme in Cyp1b1(+/+)mice. However, in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice, it restored these effects of angiotensin II. These data indicate that 6β-hydroxytestosterone contributes to increased thirst, impairment of renal function, and end-organ injury associated with angiotensin II-induced hypertension in male mice and that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension in male mice.

  12. Fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice

    SciTech Connect

    Liu, Zhi-Qin; Liu, Ting; Chen, Chuan; Li, Ming-Yan; Wang, Zi-Yu; Chen, Ruo-song; Wei, Gui-xiang; Wang, Xiao-yi; Luo, Du-Qiang

    2015-05-15

    Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathways, and its increased activity and expression are implicated in the pathogenesis of insulin resistance. Therefore, the inhibition of PTP1B is anticipated to become a potential therapeutic strategy to treat T2DM. Fumosorinone (FU), a new natural product isolated from insect fungi Isaria fumosorosea, was found to inhibit PTP1B activity in our previous study. Herein, the effects of FU on insulin resistance and mechanism in vitro and in vivo were investigated. FU increased the insulin-provoked glucose uptake in insulin-resistant HepG2 cells, and also reduced blood glucose and lipid levels of type 2 diabetic KKAy mice. FU decreased the expression of PTP1B both in insulin-resistant HepG2 cells and in liver tissues of diabetic KKAy mice. Furthermore, FU increased the phosphorylation of IRβ, IRS-2, Akt, GSK3β and Erk1/2 in insulin-resistant HepG2 cells, as well as the phosphorylation of IRβ, IRS-2, Akt in liver tissues of diabetic KKAy mice. These results showed that FU increased glucose uptake and improved insulin resistance by down-regulating the expression of PTP1B and activating the insulin signaling pathway, suggesting that it may possess antidiabetic properties. - Highlights: • Fumosorinone is a new PTP1B inhibitor isolated from insect pathogenic fungi. • Fumosorinone attenuated the insulin resistance both in vitro and in vivo. • Fumosorinone decreased the expression of PTP1B both in vitro and in vivo. • Fumosorinone activated the insulin signaling pathway both in vitro and in vivo.

  13. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish. PMID:25732591

  14. UV-B photoreceptor-mediated signalling in plants.

    PubMed

    Heijde, Marc; Ulm, Roman

    2012-04-01

    Ultraviolet-B radiation (UV-B) is a key environmental signal that is specifically perceived by plants to promote UV acclimation and survival in sunlight. Whereas the plant photoreceptors for visible light are rather well characterised, the UV-B photoreceptor UVR8 was only recently described at the molecular level. Here, we review the current understanding of the UVR8 photoreceptor-mediated pathway in the context of UV-B perception mechanism, early signalling components and physiological responses. We further outline the commonalities in UV-B and visible light signalling as well as highlight differences between these pathways.

  15. Calcium/calmodulin-mediated signal network in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2003-01-01

    Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. Calmodulin, the predominant calcium receptor, is one of the best-characterized calcium sensors in eukaryotes. In recent years, completion of the Arabidopsis genome project and advances in functional genomics have helped to identify and characterize numerous calmodulin-binding proteins in plants. There are some similarities in Ca(2+)/calmodulin-mediated signaling in plants and animals. However, plants possess multiple calmodulin genes and many calmodulin target proteins, including unique protein kinases and transcription factors. Some of these proteins are likely to act as "hubs" during calcium signal transduction. Hence, a better understanding of the function of these calmodulin target proteins should help in deciphering the Ca(2+)/calmodulin-mediated signal network and its role in plant growth, development and response to environmental stimuli.

  16. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens.

    PubMed

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection.

  17. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens

    PubMed Central

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A.

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection. PMID:26901228

  18. The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

    PubMed

    Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

    2015-11-01

    Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry.

  19. The role of stat1b in zebrafish hematopoiesis

    PubMed Central

    Song, Hao; Yan, Yi-lin; Titus, Tom; He, Xinjun; Postlethwait, John H.

    2011-01-01

    STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by subfunctionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development. PMID:21914475

  20. PACRG, a protein linked to ciliary motility, mediates cellular signaling

    PubMed Central

    Loucks, Catrina M.; Bialas, Nathan J.; Dekkers, Martijn P. J.; Walker, Denise S.; Grundy, Laura J.; Li, Chunmei; Inglis, P. Nick; Kida, Katarzyna; Schafer, William R.; Blacque, Oliver E.; Jansen, Gert; Leroux, Michel R.

    2016-01-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon–associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan. PMID:27193298

  1. PACRG, a protein linked to ciliary motility, mediates cellular signaling.

    PubMed

    Loucks, Catrina M; Bialas, Nathan J; Dekkers, Martijn P J; Walker, Denise S; Grundy, Laura J; Li, Chunmei; Inglis, P Nick; Kida, Katarzyna; Schafer, William R; Blacque, Oliver E; Jansen, Gert; Leroux, Michel R

    2016-07-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon-associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan. PMID:27193298

  2. Rac1 signaling modulates BCL-6-mediated repression of gene transcription.

    PubMed

    Barros, Patrícia; Jordan, Peter; Matos, Paulo

    2009-08-01

    Rac1 is a member of the Rho family of small GTPases that not only regulates signaling pathways involved in cell adhesion and migration but also regulates gene transcription. Here we show that the transcriptional repressor BCL-6 is regulated by Rac1 signaling. Transfection of active Rac1 mutants into colorectal DLD-1 cells led to increased expression of a BCL-6-controlled luciferase reporter construct. Conversely, inhibition of endogenous Rac1 activation by the Rac1 inhibitor NSC23766 decreased reporter activity. Moreover, BCL-6 lost its typical localization to nuclear dots upon activation of Rac1 and became predominantly soluble in a non-chromatin-bound cell fraction. Rac1 signaling also regulated the expression of endogenous BCL-6-regulated genes, including the p50 precursor NF-kappaB1/p105 and the cell adhesion molecule CD44. Interestingly, these effects were not stimulated by the alternative splice variant Rac1b. The mechanism of BCL-6 inhibition does not involve formation of a stable Rac1/BCL-6 complex and is independent of Rac-induced reactive oxygen species production or Jun NH(2)-terminal kinase activation. We show that PAK1 mediates inhibition downstream of Rac and can directly phosphorylate BCL-6. Together, these data provide substantial evidence that Rac1 signaling inhibits the transcriptional repressor BCL-6 in colorectal cells and reveal a novel pathway that links Rac1 signaling to the regulation of gene transcription.

  3. Fundamental Issues of Melatonin-Mediated Stress Signaling in Plants

    PubMed Central

    Shi, Haitao; Chen, Keli; Wei, Yunxie; He, Chaozu

    2016-01-01

    As a widely known hormone in animals, melatonin (N-acetyl-5-methoxytryptamine) has been more and more popular research topic in various aspects of plants. To summarize the these recent advances, this review focuses on the regulatory effects of melatonin in plant response to multiple abiotic stresses including salt, drought, cold, heat and oxidative stresses and biotic stress such as pathogen infection. We highlight the changes of endogenous melatonin levels under stress conditions, and the extensive metabolome, transcriptome, and proteome reprogramming by exogenous melatonin application. Moreover, melatonin-mediated stress signaling and underlying mechanism in plants are extensively discussed. Much more is needed to further study in detail the mechanisms of melatonin-mediated stress signaling in plants. PMID:27512404

  4. Fundamental Issues of Melatonin-Mediated Stress Signaling in Plants.

    PubMed

    Shi, Haitao; Chen, Keli; Wei, Yunxie; He, Chaozu

    2016-01-01

    As a widely known hormone in animals, melatonin (N-acetyl-5-methoxytryptamine) has been more and more popular research topic in various aspects of plants. To summarize the these recent advances, this review focuses on the regulatory effects of melatonin in plant response to multiple abiotic stresses including salt, drought, cold, heat and oxidative stresses and biotic stress such as pathogen infection. We highlight the changes of endogenous melatonin levels under stress conditions, and the extensive metabolome, transcriptome, and proteome reprogramming by exogenous melatonin application. Moreover, melatonin-mediated stress signaling and underlying mechanism in plants are extensively discussed. Much more is needed to further study in detail the mechanisms of melatonin-mediated stress signaling in plants. PMID:27512404

  5. Transposon-Mediated Alteration of TaMATE1B Expression in Wheat Confers Constitutive Citrate Efflux from Root Apices[W

    PubMed Central

    Tovkach, Andriy; Ryan, Peter R.; Richardson, Alan E.; Lewis, David C.; Rathjen, Tina M.; Ramesh, Sunita; Tyerman, Stephen D.; Delhaize, Emmanuel

    2013-01-01

    The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3′ regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance. PMID:23204428

  6. Root signals that mediate mutualistic interactions in the rhizosphere.

    PubMed

    Rasmann, Sergio; Turlings, Ted Cj

    2016-08-01

    A recent boom in research on belowground ecology is rapidly revealing a multitude of fascinating interactions, in particular in the rhizosphere. Many of these interactions are mediated by photo-assimilates that are excreted by plant roots. Root exudates are not mere waste products, but serve numerous functions to control abiotic and biotic processes. These functions range from changing the chemical and physical properties of the soil, inhibiting the growth of competing plants, combatting herbivores, and regulating the microbial community. Particularly intriguing are root-released compounds that have evolved to serve mutualistic interactions with soil-dwelling organisms. These mutually beneficial plant-mediated signals are not only of fundamental ecological interest, but also exceedingly important from an agronomical perspective. Here, we attempt to provide an overview of the plant-produced compounds that have so far been implicated in mutualistic interactions. We propose that these mutualistic signals may have evolved from chemical defenses and we point out that they can be (mis)used by specialized pathogens and herbivores. We speculate that many more signals and interactions remain to be uncovered and that a good understanding of the mechanisms and ecological implications can be the basis for exploitation and manipulation of the signals for crop improvement and protection. PMID:27393937

  7. The mechanisms of HAMP-mediated signaling in transmembrane receptors.

    PubMed

    Ferris, Hedda U; Dunin-Horkawicz, Stanislaw; Mondéjar, Laura García; Hulko, Michael; Hantke, Klaus; Martin, Jörg; Schultz, Joachim E; Zeth, Kornelius; Lupas, Andrei N; Coles, Murray

    2011-03-01

    HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.

  8. LIG4 mediates Wnt signalling-induced radioresistance

    PubMed Central

    Jun, Sohee; Jung, Youn-Sang; Suh, Han Na; Wang, Wenqi; Kim, Moon Jong; Oh, Young Sun; Lien, Esther M.; Shen, Xi; Matsumoto, Yoshihisa; McCrea, Pierre D.; Li, Lei; Chen, Junjie; Park, Jae-Il

    2016-01-01

    Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of β-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by β-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/β-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with β-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration. PMID:27009971

  9. LIG4 mediates Wnt signalling-induced radioresistance.

    PubMed

    Jun, Sohee; Jung, Youn-Sang; Suh, Han Na; Wang, Wenqi; Kim, Moon Jong; Oh, Young Sun; Lien, Esther M; Shen, Xi; Matsumoto, Yoshihisa; McCrea, Pierre D; Li, Lei; Chen, Junjie; Park, Jae-Il

    2016-01-01

    Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of β-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by β-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/β-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with β-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration. PMID:27009971

  10. HNF1B controls proximal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2.

    PubMed

    Heliot, Claire; Desgrange, Audrey; Buisson, Isabelle; Prunskaite-Hyyryläinen, Renata; Shan, Jingdong; Vainio, Seppo; Umbhauer, Muriel; Cereghini, Silvia

    2013-02-01

    The nephron is a highly specialised segmented structure that provides essential filtration and resorption renal functions. It arises by formation of a polarised renal vesicle that differentiates into a comma-shaped body and then a regionalised S-shaped body (SSB), with the main prospective segments mapped to discrete domains. The regulatory circuits involved in initial nephron patterning are poorly understood. We report here that HNF1B, a transcription factor known to be involved in ureteric bud branching and initiation of nephrogenesis, has an additional role in segment fate acquisition. Hnf1b conditional inactivation in murine nephron progenitors results in rudimentary nephrons comprising a glomerulus connected to the collecting system by a short tubule displaying distal fates. Renal vesicles develop and polarise normally but fail to progress to correctly patterned SSBs. Major defects are evident at late SSBs, with altered morphology, reduction of a proximo-medial subdomain and increased apoptosis. This is preceded by strong downregulation of the Notch pathway components Lfng, Dll1 and Jag1 and the Irx1/2 factors, which are potential regulators of proximal and Henle's loop segment fates. Moreover, HNF1B is recruited to the regulatory sequences of most of these genes. Overexpression of a HNF1B dominant-negative construct in Xenopus embryos causes downregulation specifically of proximal and intermediate pronephric segment markers. These results show that HNF1B is required for the acquisition of a proximo-intermediate segment fate in vertebrates, thus uncovering a previously unappreciated function of a novel SSB subcompartment in global nephron segmentation and further differentiation.

  11. Montelukast Disposition: No Indication of Transporter-Mediated Uptake in OATP2B1 and OATP1B1 Expressing HEK293 Cells

    PubMed Central

    Brännström, Marie; Nordell, Pär; Bonn, Britta; Davis, Andrew M.; Palmgren, Anna-Pia; Hilgendorf, Constanze; Rubin, Katarina; Grime, Ken

    2015-01-01

    Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast. PMID:26694455

  12. Montelukast Disposition: No Indication of Transporter-Mediated Uptake in OATP2B1 and OATP1B1 Expressing HEK293 Cells.

    PubMed

    Brännström, Marie; Nordell, Pär; Bonn, Britta; Davis, Andrew M; Palmgren, Anna-Pia; Hilgendorf, Constanze; Rubin, Katarina; Grime, Ken

    2015-01-01

    Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast. PMID:26694455

  13. Negative Regulation of Cytoplasmic RNA-Mediated Antiviral Signaling

    PubMed Central

    Komuro, Akihiko; Bamming, Darja

    2008-01-01

    The recent, rapid progress in our understanding of cytoplasmic RNA-mediated antiviral innate immune signaling was initiated by the discovery of retinoic acid-inducible gene I (RIG-I) as a sensor of viral RNA [1]. It is now widely recognized that RIG-I and related RNA helicases, melanoma differentiated-associated gene-5 (MDA5) and laboratory of genetics and physiology-2 (LGP2), can initiate and/or regulate RNA and virus -mediated type I IFN production and antiviral responses. As with other cytokine systems, production of type I IFN is a transient process, and can be hazardous to the host if unregulated, resulting in chronic cellular toxicity or inflammatory and autoimmune diseases [2-9]. In addition, the RIG-I-like receptor (RLR) system is a fundamental target for virus-encoded immune suppression, with many indirect and direct examples of interference described. In this article, we review the current understanding of endogenous negative regulation in RLR signaling and explore direct inhibition of RLR signaling by viruses as a host immune evasion strategy. PMID:18703349

  14. Zinc ferrite nanoparticles activate IL-1b, NFKB1, CCL21 and NOS2 signaling to induce mitochondrial dependent intrinsic apoptotic pathway in WISH cells

    SciTech Connect

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ahmad, Javed; Siddiqui, Maqsood A.; Dwivedi, Sourabh; Khan, Shams T.; Musarrat, Javed

    2013-12-01

    The present study has demonstrated the translocation of zinc ferrite nanoparticles (ZnFe{sub 2}O{sub 4}-NPs) into the cytoplasm of human amnion epithelial (WISH) cells, and the ensuing cytotoxicity and genetic damage. The results suggested that in situ NPs induced oxidative stress, alterations in cellular membrane and DNA strand breaks. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and neutral red uptake (NRU) cytotoxicity assays indicated 64.48 ± 1.6% and 50.73 ± 2.1% reduction in cell viability with 100 μg/ml of ZnFe{sub 2}O{sub 4}-NPs exposure. The treated WISH cells exhibited 1.2-fold higher ROS level with 0.9-fold decline in membrane potential (ΔΨm) and 7.4-fold higher DNA damage after 48 h of ZnFe{sub 2}O{sub 4}-NPs treatment. Real-time PCR (qPCR) analysis of p53, CASP 3 (caspase-3), and bax genes revealed 5.3, 1.6, and 14.9-fold upregulation, and 0.18-fold down regulation of bcl 2 gene vis-à-vis untreated control. RT{sup 2} Profiler™ PCR array data elucidated differential up-regulation of mRNA transcripts of IL-1b, NFKB1, NOS2 and CCL21 genes in the range of 1.5 to 3.7-folds. The flow cytometry based cell cycle analysis suggested the transfer of 15.2 ± 2.1% (p < 0.01) population of ZnFe{sub 2}O{sub 4}-NPs (100 μg/ml) treated cells into apoptotic phase through intrinsic pathway. Over all, the data revealed the potential of ZnFe{sub 2}O{sub 4}-NPs to induce cellular and genetic toxicity in cells of placental origin. Thus, the significant ROS production, reduction in ΔΨm, DNA damage, and activation of genes linked to inflammation, oxidative stress, proliferation, DNA damage and repair could serve as the predictive toxicity and stress markers for ecotoxicological assessment of ZnFe{sub 2}O{sub 4}-NPs induced cellular and genetic damage. - Highlights: • First report on the molecular toxicity of ZnFe{sub 2}O{sub 4}-NPs in cells of placental origin • WISH cells treated with ZnFe{sub 2}O{sub 4}-NPs exhibited cytoplasmic

  15. Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1

    PubMed Central

    Feng, Mingxiao; Kim, Jae-Yean

    2015-01-01

    It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) (SCFTIR1/AFB) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional SCFTIR1/AFB auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research. PMID:26467289

  16. Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1.

    PubMed

    Feng, Mingxiao; Kim, Jae-Yean

    2015-10-01

    It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) (SCF(TIR1/AFB)) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional SCF(TIR1/AFB) auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research.

  17. Integrating Hormone- and Micromolecule-Mediated Signaling with Plasmodesmal Communication.

    PubMed

    Han, Xiao; Kim, Jae-Yean

    2016-01-01

    Intercellular and supracellular communications through plasmodesmata are involved in vital processes for plant development and physiological responses. Micro- and macromolecules, including hormones, RNA, and proteins, serve as biological information vectors that traffic through the plasmodesmata between cells. Previous studies demonstrated that the plasmodesmata are elaborately regulated, whereby a long queue of multiple signaling molecules forms. However, the mechanism by which these signals are coupled or coordinated in terms of simultaneous transport in a single channel remains a puzzle. In the last few years, several phytohormones that could function as both non-cell-autonomous signals and plasmodesmal regulators have been disclosed. Plasmodesmal regulators such as auxin, salicylic acid, reactive oxygen species, gibberellic acids, chitin, and jasmonic acid could regulate intercellular trafficking by adjusting plasmodesmal permeability. Here, callose, along with β-glucan synthase and β-glucanase, plays a critical role in regulating plasmodesmal permeability. Interestingly, most of the previously identified regulators are capable of diffusing through the plasmodesmata. Given the small sizes of these molecules, the plasmodesmata are prominent intercellular channels that allow diffusion-based movement of those signaling molecules. Obviously, intercellular communication is under the control of a major mechanism, named a feedback loop, at the plasmodesmata, which mediates complicated biological behaviors. Prospective research on the mechanism of coupling micromolecules at the plasmodesmata for developmental signaling and nutrient provision will help us to understand how plants coordinate their development and photosynthetic assimilation, which is important for agriculture.

  18. Reactive oxygen species mediate insulin signal transduction in mouse hypothalamus.

    PubMed

    Onoue, Takeshi; Goto, Motomitsu; Tominaga, Takashi; Sugiyama, Mariko; Tsunekawa, Taku; Hagiwara, Daisuke; Banno, Ryoichi; Suga, Hidetaka; Sugimura, Yoshihisa; Arima, Hiroshi

    2016-04-21

    In the hypothalamus, several reports have implied that ROS mediate physiological effects of insulin. In this study, we investigated the mechanisms of insulin-induced ROS production and the effect of ROS on insulin signal transduction in mouse hypothalamic organotypic cultures. Insulin increased intracellular ROS, which were suppressed by NADPH oxidase inhibitor. H2O2 increased phospho-insulin receptor β (p-IRβ) and phospho-Akt (p-Akt) levels. Insulin-induced increases in p-IRβ and p-Akt levels were attenuated by ROS scavenger or NADPH oxidase inhibitor. Our data suggest that insulin-induced phosphorylation of IRβ and Akt is mediated via ROS which are predominantly produced by NADPH oxidase in mouse hypothalamus.

  19. A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

    PubMed

    Patterson, Heide Christine; Gerbeth, Carolin; Thiru, Prathapan; Vögtle, Nora F; Knoll, Marko; Shahsafaei, Aliakbar; Samocha, Kaitlin E; Huang, Cher X; Harden, Mark Michael; Song, Rui; Chen, Cynthia; Kao, Jennifer; Shi, Jiahai; Salmon, Wendy; Shaul, Yoav D; Stokes, Matthew P; Silva, Jeffrey C; Bell, George W; MacArthur, Daniel G; Ruland, Jürgen; Meisinger, Chris; Lodish, Harvey F

    2015-10-20

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.

  20. A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling

    PubMed Central

    Patterson, Heide Christine; Gerbeth, Carolin; Thiru, Prathapan; Vögtle, Nora F.; Knoll, Marko; Shahsafaei, Aliakbar; Samocha, Kaitlin E.; Huang, Cher X.; Harden, Mark Michael; Song, Rui; Chen, Cynthia; Kao, Jennifer; Shi, Jiahai; Salmon, Wendy; Shaul, Yoav D.; Stokes, Matthew P.; Silva, Jeffrey C.; Bell, George W.; MacArthur, Daniel G.; Ruland, Jürgen; Meisinger, Chris; Lodish, Harvey F.

    2015-01-01

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells. PMID:26438848

  1. Contrasting signaling pathways of alpha1A- and alpha1B-adrenergic receptor subtype activation of phosphatidylinositol 3-kinase and Ras in transfected NIH3T3 cells.

    PubMed

    Hu, Z W; Shi, X Y; Lin, R Z; Hoffman, B B

    1999-01-01

    Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including alpha1-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human alpha1-receptors, namely, alpha1A, alpha1B and alpha1D to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human alpha1A and alpha1B via pertussis toxin-insensitive G proteins; alpha1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with the alpha-subunit of the G protein transducin (alpha(t)) a scavenger of betagamma-subunits released from activated G proteins, inhibited alpha1B-receptor but not alpha1A-receptor-stimulated PI 3-kinase activity. Stimulation of both alpha1A- and alpha1B-receptors activated p21ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21ras attenuated alpha1B-receptor but not alpha1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuated alpha1A- but not alpha1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of the alpha1-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

  2. Growth hormone STAT5-mediated signaling and its modulation in mice liver during the growth period.

    PubMed

    Martinez, Carolina S; Piazza, Verónica G; Ratner, Laura D; Matos, Marina N; González, Lorena; Rulli, Susana B; Miquet, Johanna G; Sotelo, Ana I

    2013-01-01

    Postnatal growth exhibits two instances of rapid growth in mice: the first is perinatal and independent of growth hormone (GH), the second is peripuberal and GH-dependent. Signal transducer and activator of transcription 5b (STAT5b) is the main GH-signaling mediator and it is related to IGF1 synthesis and somatic growth. The aim of this work was to assess differential STAT5 sensitivity to GH during the growth period in mouse liver of both sexes. Three representative ages were selected: 1-week-old animals, in the GH-independent phase of growth; 2.5-week-old mice, at the onset of the GH-dependent phase of growth; and 9-week-old young adults. GH-signaling mediators were assessed by immunoblotting, quantitative RT-PCR and immunohistochemistry. GH-induced STAT5 phosphorylation is low at one-week and maximal at 2.5-weeks of age when compared to young adults, accompanied by higher protein content at the onset of growth. Suppressor CIS and phosphatase PTP1B exhibit high levels in one-week animals, which gradually decline, while SOCS2 and SOCS3 display higher levels at adulthood. Nuclear phosphorylated STAT5 is low in one-week animals while in 2.5-week animals it is similar to 9-week control; expression of SOCS3, an early response GH-target gene, mimics this pattern. STAT5 coactivators glucocorticoid receptor (GR) and hepatic nuclear factor 1 (HNF1) abundance is higher in adulthood. Therefore, GH-induced STAT5 signaling presents age-dependent activity in liver, with its maximum coinciding with the onset of GH-dependent phase of growth, accompanied by an age-dependent variation of modulating factors. This work contributes to elucidate the molecular mechanisms implicated in GH responsiveness during growth.

  3. Signal Transduction Model of Magnetic Sensing in Cryptochrome Mediated Photoreception

    NASA Astrophysics Data System (ADS)

    Todd, Phillise Tiffeny

    While migratory birds have long been known to use the Earth's magnetic field for navigation, the precise biophysical mechanism behind this magnetic sense remains unconfirmed. A leading theory of magnetoreception suggests a chemical compass model with a yet undetermined molecular reaction site and unknown magnetically sensitive reactants. The cryptochrome photoreceptor has emerged as a promising candidate site. This investigation numerically models the first order kinetics of cryptochrome mediated photoreception, in order to evaluate its ability to function as a magnetic sensor and transduce orientation information along a neural pathway. A signal-to-noise ratio is defined to quantify the threshold for the functioning of a cryptochrome-based chemical compass. The model suggests that a flavin-superoxide radical pair in cryptochrome functions as the chemical reactants for magnetoreception. Such a cryptochrome-based signal transduction model reasonably predicts the general light intensity and wavelength effects that have been experimentally observed in migratory birds.

  4. Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

    2013-03-01

    Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

  5. Calcium signaling mediates cold sensing in insect tissues.

    PubMed

    Teets, Nicholas M; Yi, Shu-Xia; Lee, Richard E; Denlinger, David L

    2013-05-28

    The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

  6. Calcium signaling mediates cold sensing in insect tissues

    PubMed Central

    Teets, Nicholas M.; Yi, Shu-Xia; Lee, Richard E.; Denlinger, David L.

    2013-01-01

    The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms. PMID:23671084

  7. Calcium signaling mediates cold sensing in insect tissues.

    PubMed

    Teets, Nicholas M; Yi, Shu-Xia; Lee, Richard E; Denlinger, David L

    2013-05-28

    The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms. PMID:23671084

  8. Proposed glucocorticoid-mediated zinc signaling in the hippocampus.

    PubMed

    Takeda, Atsushi; Tamano, Haruna

    2012-07-01

    Corticosteroid hormones are secreted from the adrenal glands in hourly pulses and signal the hippocampus for the development and function. In contrast, the stress-induced rise in corticosteroid concentrations has a profound effect on emotional arousal, motivational processes and cognitive performance. This rise is required as the stress response to maintain homeostasis in the living body or restore it. However, abnormal rise in corticosteroid concentrations is a disadvantage to the hippocampus. Corticosteroid-glutamatergic interactions during information processing are proposed as a potential model to explain many of the diverse actions of corticosteroids in synaptic plasticity such as long-term potentiation and cognition. Because zincergic neurons are a subtype of glutamatergic neurons and release Zn(2+) and glutamate into the synaptic cleft, it is possible that homeostasis of synaptic Zn(2+), in addition to homeostasis of glutamate, is modified by glucocorticoids, followed by the changes in cognitive function and stress response. Zn(2+) signal participates in cognitive and emotional behavior in cooperation with signaling of glucocorticoids and glutamate, while can disadvantageously act on the hippocampus under sever stress circumstances. This paper analyzes the actions of glucocorticoid-mediated Zn(2+) signal in the hippocampus under stressful circumstances and its significance in both hippocampal function and dysfunction.

  9. CD99 inhibits CD98-mediated β1 integrin signaling through SHP2-mediated FAK dephosphorylation.

    PubMed

    Lee, Kyoung Jin; Yoo, Yeon Ho; Kim, Min Seo; Yadav, Birendra Kumar; Kim, Yuri; Lim, Dongyoung; Hwangbo, Cheol; Moon, Ki Won; Kim, Daejoong; Jeoung, Dooil; Lee, Hansoo; Lee, Jeong-Hyung; Hahn, Jang-Hee

    2015-08-15

    The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein. It has been previously shown that CD99 and CD98 oppositely regulate β1 integrin signaling, though the mechanisms by which this regulation occurs are not known. Our results revealed that antibody-mediated crosslinking of CD98 induced FAK phosphorylation at Y397 and facilitated the formation of the protein kinase Cα (PKCα)-syntenin-focal adhesion kinase (FAK), focal adhesions (FAs), and IPP-Akt1-syntenin complex, which mediates β1 integrin signaling. In contrast, crosslinking of CD99 disrupted the formation of the PKCα-syntenin-FAK complex as well as FA via FAK dephosphorylation. The CD99-induced dephosphorylation of FAK was apparently mediated by the recruitment of Src homology region 2 domain-containing phosphatase-2 (SHP2) to the plasma membrane and subsequent activation of its phosphatase activity. Further consequences of the activation of SHP2 included the disruption of FAK-talin and talin-β1 integrin interactions and attenuation in the formation of the IPP-Akt1-syntenin complex at the plasma membrane, which resulted in reduced cell-ECM adhesion. This report uncovers the molecular mechanisms underlying the inverse regulation of β1 integrin signaling by CD99 and CD98 and may provide a novel therapeutic approach to treat inflammation and cancer.

  10. Hippocampus ghrelin signaling mediates appetite through lateral hypothalamic orexin pathways

    PubMed Central

    Hsu, Ted M; Hahn, Joel D; Konanur, Vaibhav R; Noble, Emily E; Suarez, Andrea N; Thai, Jessica; Nakamoto, Emily M; Kanoski, Scott E

    2015-01-01

    Feeding behavior rarely occurs in direct response to metabolic deficit, yet the overwhelming majority of research on the biology of food intake control has focused on basic metabolic and homeostatic neurobiological substrates. Most animals, including humans, have habitual feeding patterns in which meals are consumed based on learned and/or environmental factors. Here we illuminate a novel neural system regulating higher-order aspects of feeding through which the gut-derived hormone ghrelin communicates with ventral hippocampus (vHP) neurons to stimulate meal-entrained conditioned appetite. Additional results show that the lateral hypothalamus (LHA) is a critical downstream substrate for vHP ghrelin-mediated hyperphagia and that vHP ghrelin activated neurons communicate directly with neurons in the LHA that express the neuropeptide, orexin. Furthermore, activation of downstream orexin-1 receptors is required for vHP ghrelin-mediated hyperphagia. These findings reveal novel neurobiological circuitry regulating appetite through which ghrelin signaling in hippocampal neurons engages LHA orexin signaling. DOI: http://dx.doi.org/10.7554/eLife.11190.001 PMID:26745307

  11. Hippocampus ghrelin signaling mediates appetite through lateral hypothalamic orexin pathways.

    PubMed

    Hsu, Ted M; Hahn, Joel D; Konanur, Vaibhav R; Noble, Emily E; Suarez, Andrea N; Thai, Jessica; Nakamoto, Emily M; Kanoski, Scott E

    2015-12-15

    Feeding behavior rarely occurs in direct response to metabolic deficit, yet the overwhelming majority of research on the biology of food intake control has focused on basic metabolic and homeostatic neurobiological substrates. Most animals, including humans, have habitual feeding patterns in which meals are consumed based on learned and/or environmental factors. Here we illuminate a novel neural system regulating higher-order aspects of feeding through which the gut-derived hormone ghrelin communicates with ventral hippocampus (vHP) neurons to stimulate meal-entrained conditioned appetite. Additional results show that the lateral hypothalamus (LHA) is a critical downstream substrate for vHP ghrelin-mediated hyperphagia and that vHP ghrelin activated neurons communicate directly with neurons in the LHA that express the neuropeptide, orexin. Furthermore, activation of downstream orexin-1 receptors is required for vHP ghrelin-mediated hyperphagia. These findings reveal novel neurobiological circuitry regulating appetite through which ghrelin signaling in hippocampal neurons engages LHA orexin signaling.

  12. Mitotic lamin disassembly is triggered by lipid-mediated signaling.

    PubMed

    Mall, Moritz; Walter, Thomas; Gorjánácz, Mátyás; Davidson, Iain F; Nga Ly-Hartig, Thi Bach; Ellenberg, Jan; Mattaj, Iain W

    2012-09-17

    Disassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event.

  13. Sumatriptan inhibition of N-type calcium channel mediated signaling in dural CGRP terminal fibres

    PubMed Central

    Baillie, Landon D.; Ahn, Andrew H.; Mulligan, Sean J.

    2012-01-01

    The selective 5-HT1 receptor agonist sumatriptan is an effective therapeutic for migraine pain yet the antimigraine mechanisms of action remain controversial. Pain-responsive fibres containing calcitonin gene-related peptide (CGRP) densely innervating the cranial dura mater are widely believed to be an essential anatomical substrate for the development of migraine pain. 5HT1 receptors in the dura colocalize with CGRP fibres in high density and thus provide a possible peripheral site of action for sumatriptan. In the present study, we used high-resolution optical imaging selectively within individual mouse dural CGRP nociceptive fibre terminations and found that application of sumatriptan caused a rapid, reversible dose-dependent inhibition in the amplitude of single action potential evoked Ca2+ transients. Pre-application of the 5-HT1 antagonist GR127935 or the selective 5-HT1D antagonist BRL 15572 prevented inhibition while the selective 5-HT1B antagonist SB 224289 did not, suggesting this effect was mediated selectively through the 5-HT1D receptor subtype. Sumatriptan inhibition of the action potential evoked Ca2+ signaling was mediated selectively through N-type Ca2+ channels. Although the T-type Ca2+ channel accounted for a greater proportion of the Ca2+ signal it did not mediate any of the sumatriptan inhibition. Our findings support a peripheral site of action for sumatriptan in inhibiting the activity of dural pain fibres selectively through a single Ca2+ channel subtype. This finding adds to our understanding of the mechanisms that underlie the clinical effectiveness of 5HT1 receptor agonists such as sumatriptan and may provide insight for the development of novel peripherally targeted therapeutics for mitigating the pain of migraine. PMID:22691374

  14. The involvement of intracellular Ca2+ in 5-HT1B/1D receptor-mediated contraction of the rabbit isolated renal artery

    PubMed Central

    Hill, P B; Dora, K A; Hughes, A D; Garland, C J

    2000-01-01

    5-Hydroxytryptamine1B/1D (5-HT1B/1D) receptor coupling to contraction was investigated in endothelium-denuded rabbit isolated renal arteries, by simultaneously measuring tension and intracellular [Ca2+], and tension in permeabilized smooth muscle cells.In intact arterial segments, 1 nM–10 μM 5-HT failed to induce contraction or increase the fura-2 fluorescence ratio (in the presence of 1 μM ketanserin and prazosin to block 5-HT2 and α1-adrenergic receptors, respectively). However, in vessels pre-exposed to either 20 mM K+ or 30 nM U46619, 5-HT stimulated concentration-dependent increases in both tension and intracellular [Ca2+].1 nM–10 μM U46619 induced concentration-dependent contractions. In the presence of nifedipine (0.3 and 1 μM) the maximal contraction to U46619 (10 μM) was reduced by around 70%. The residual contraction was abolished by the putative receptor operated channel inhibitor, SKF 96365 (2 μM).With 0.3 μM nifedipine present, 100 nM U46619 evoked similar contraction to 30 nM U46619 in the absence of nifedipine, but contraction to 5-HT (1 nM–10 μM) was abolished.In permeabilized arterial segments, 10 mM caffeine, 1 μM IP3 or 100 μM phenylephrine, each evoked transient contractions by releasing Ca2+ from intracellular stores, whereas 5-HT had no effect. In intact arterial segments pre-stimulated with 20 mM K+, 5-HT-evoked contractions were unaffected by 1 μM thapsigargin, which inhibits sarco- and endoplasmic reticulum calcium-ATPases.In vessels permeabilized with α-toxin and then pre-contracted with Ca2+ and GTP, 5-HT evoked further contraction, reflecting increased myofilament Ca2+-sensitivity.Contraction linked to 5-HT1B/1D receptor stimulation in the rabbit renal artery can be explained by an influx of external Ca2+ through voltage-dependent Ca2+ channels and sensitization of the contractile myofilaments to existing levels of Ca2+, with no release of Ca2+ from intracellular stores. PMID

  15. CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement

    PubMed Central

    Ju, Wujian; Yang, Sihyung; Ansede, John H.; Stephens, Chad E.; Bridges, Arlene S.; Voyksner, Robert D.; Ismail, Mohamed A.; Boykin, David W.; Tidwell, Richard R.; Hall, James Edwin; Wang, Michael Zhuo

    2013-01-01

    DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine if differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of an oxygen into the amidine C=N to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of nitric oxide. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules. PMID:24186380

  16. Chaperone-mediated specificity in Ras and Rap signaling.

    PubMed

    Azoulay-Alfaguter, Inbar; Strazza, Marianne; Mor, Adam

    2015-01-01

    Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.

  17. The expanding regulatory network of STING-mediated signaling.

    PubMed

    Surpris, Guy; Poltorak, Alexander

    2016-08-01

    The identification and characterization of DNA-sensing pathways has been a subject of intensive investigation for the last decade. This interest, in part, is supported by the fact that the main outcome of DNA-responses is production of type I interferon (IFN-I), which, if produced in excessive amounts, leads to various pathologies. STING (stimulator of interferon genes) is positioned in the center of these responses and is activated either via direct sensing of second messengers or via interaction with upstream sensors of dsDNA. STING mediates responses to pathogens as well as host-derived DNA and is, therefore, linked to various autoimmune diseases, cancer predisposition and ageing. Recent mouse models of DNA damage showed the adaptor STING to be crucial for heightened resting levels of IFN-I. In this review, we will focus on recent advances in understanding the regulation of STING-signaling and identification of its novel components. PMID:27414485

  18. Molecular architecture of endocannabinoid signaling at nociceptive synapses mediating analgesia.

    PubMed

    Nyilas, Rita; Gregg, Laura C; Mackie, Ken; Watanabe, Masahiko; Zimmer, Andreas; Hohmann, Andrea G; Katona, István

    2009-05-01

    Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid-mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called 2-arachidonoylglycerol (2-AG) is mobilized in the lumbar spinal cord in temporal correlation with stress-induced antinociception. We therefore characterized the precise molecular architecture of 2-AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase-alpha (DGL-alpha), the synthesizing enzyme of 2-AG. Peroxidase-based immunocytochemistry demonstrated high levels of DGL-alpha protein and CB(1) cannabinoid receptor, a receptor for 2-AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High-resolution electron microscopy uncovered postsynaptic localization of DGL-alpha at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB(1) on excitatory axon terminals. Furthermore, DGL-alpha in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR(5)), whose activation induces 2-AG biosynthesis. Finally, intrathecal activation of mGluR(5) at the lumbar level evoked endocannabinoid-mediated stress-induced analgesia through the DGL-2-AG-CB(1) pathway. Taken together, these findings suggest a key role for 2-AG-mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of 2-AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal 2-AG levels may be an efficacious way to regulate pain

  19. Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research

    PubMed Central

    Li, Ning; Hong, Tianqi; Li, Rong; Wang, Yao; Guo, Mengjiao; Cao, Zongxi; Cai, Yumei; Liu, Sidang; Chai, Tongjie; Wei, Liangmeng

    2016-01-01

    Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks. PMID:27708647

  20. Endocannabinoid signaling mediates oxytocin-driven social reward

    PubMed Central

    Wei, Don; Lee, DaYeon; Cox, Conor D.; Karsten, Carley A.; Peñagarikano, Olga; Geschwind, Daniel H.; Gall, Christine M.; Piomelli, Daniele

    2015-01-01

    Marijuana exerts profound effects on human social behavior, but the neural substrates underlying such effects are unknown. Here we report that social contact increases, whereas isolation decreases, the mobilization of the endogenous marijuana-like neurotransmitter, anandamide, in the mouse nucleus accumbens (NAc), a brain structure that regulates motivated behavior. Pharmacological and genetic experiments show that anandamide mobilization and consequent activation of CB1 cannabinoid receptors are necessary and sufficient to express the rewarding properties of social interactions, assessed using a socially conditioned place preference test. We further show that oxytocin, a neuropeptide that reinforces parental and social bonding, drives anandamide mobilization in the NAc. Pharmacological blockade of oxytocin receptors stops this response, whereas chemogenetic, site-selective activation of oxytocin neurons in the paraventricular nucleus of the hypothalamus stimulates it. Genetic or pharmacological interruption of anandamide degradation offsets the effects of oxytocin receptor blockade on both social place preference and cFos expression in the NAc. The results indicate that anandamide-mediated signaling at CB1 receptors, driven by oxytocin, controls social reward. Deficits in this signaling mechanism may contribute to social impairment in autism spectrum disorders and might offer an avenue to treat these conditions. PMID:26504214

  1. Luminal Ca2+ dynamics during IP3R mediated signals

    NASA Astrophysics Data System (ADS)

    Lopez, Lucia F.; Ponce Dawson, Silvina

    2016-06-01

    The role of cytosolic Ca2+ on the kinetics of Inositol 1,4,5-triphosphate receptors (IP3Rs) and on the dynamics of IP3R-mediated Ca2+ signals has been studied at large both experimentally and by modeling. The role of luminal Ca2+ has not been investigated with that much detail although it has been found that it is relevant for signal termination in the case of Ca2+ release through ryanodine receptors. In this work we present the results of observing the dynamics of luminal and cytosolic Ca2+ simultaneously in Xenopus laevis oocytes. Combining observations and modeling we conclude that there is a rapid mechanism that guarantees the availability of free Ca2+ in the lumen even when a relatively large Ca2+ release is evoked. Comparing the dynamics of cytosolic and luminal Ca2+ during a release, we estimate that they are consistent with a 80% of luminal Ca2+ being buffered. The rapid availability of free luminal Ca2+ correlates with the observation that the lumen occupies a considerable volume in several regions across the images.

  2. DELLA-mediated gibberellin signalling regulates Nod factor signalling and rhizobial infection

    PubMed Central

    Fonouni-Farde, Camille; Tan, Sovanna; Baudin, Maël; Brault, Mathias; Wen, Jiangqi; Mysore, Kirankumar S.; Niebel, Andreas; Frugier, Florian; Diet, Anouck

    2016-01-01

    Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections. PMID:27586842

  3. DELLA-mediated gibberellin signalling regulates Nod factor signalling and rhizobial infection.

    PubMed

    Fonouni-Farde, Camille; Tan, Sovanna; Baudin, Maël; Brault, Mathias; Wen, Jiangqi; Mysore, Kirankumar S; Niebel, Andreas; Frugier, Florian; Diet, Anouck

    2016-01-01

    Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections. PMID:27586842

  4. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

    PubMed Central

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  5. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

  6. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  7. Triton 2 (1B)

    NASA Technical Reports Server (NTRS)

    Clark, Michelle L.; Meiss, A. G.; Neher, Jason R.; Rudolph, Richard H.

    1994-01-01

    The goal of this project was to perform a detailed design analysis on a conceptually designed preliminary flight trainer. The Triton 2 (1B) must meet the current regulations in FAR Part 23. The detailed design process included the tasks of sizing load carrying members, pulleys, bolts, rivets, and fuselage skin for the safety cage, empennage, and control systems. In addition to the regulations in FAR Part 23, the detail design had to meet established minimums for environmental operating conditions and material corrosion resistance.

  8. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    SciTech Connect

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  9. Characterization of NAADP-mediated calcium signaling in human spermatozoa

    SciTech Connect

    Sánchez-Tusie, A.A.; Vasudevan, S.R.; Churchill, G.C.; Nishigaki, T.; Treviño, C.L.

    2014-01-10

    Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca{sup 2+}]{sub i} increases in human sperm in the absence of [Ca{sup 2+}]{sub o}. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca{sup 2+} signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca{sup 2+}-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca{sup 2+} signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca{sup 2+} and pH. Ca{sup 2+} fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca{sup 2+}] increases in human sperm even in the absence of extracellular Ca{sup 2+}. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.

  10. [Rho-mediated signal transduction and its physiological roles].

    PubMed

    Ishizaki, Toshimasa

    2003-03-01

    Rho is a member of the Ras-related family of small molecular weight GTP-binding proteins, and Rho works as a molecular switch by shuttling between the GDP-bound inactive form and the GTP-bound active form. Rho is involved in cell motility, cell adhesion, and cytokinesis through the reorganization of the actin cytoskeleton. In addition to this, Rho also regulates Ras-induced transformation, transcriptional activation and cell cycle progression. These actions through the Rho signaling are mediated by downstream Rho effectors. Several putative Rho effectors including ROCK and mDia have been isolated on the basis of their selective binding to the GTP-bound form of Rho. Among them, the ROCK family of Rho-associated serine/threonine protein kinases inactivates myosin phosphatase and actin depolymerizing factor (cofilin/Destrin) to induce stabilization of filamentous actin and increase in the actomyosin-based contractility. mDia binds profilin likely to promote actin polymerization. Thus, these effectors are supposed to work in organization of the actin cytoskeleton. Furthermore, analyses using a ROCK specific inhibitor Y-27632 have suggested that the Rho-ROCK pathway works in contractions of vascular smooth muscles and is involved in malignant cell transformation and tumor invasion and metastasis.

  11. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    SciTech Connect

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-12-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  12. CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis

    PubMed Central

    Miller, Rachel E.; Tran, Phuong B.; Das, Rosalina; Ghoreishi-Haack, Nayereh; Ren, Dongjun; Miller, Richard J.; Malfait, Anne-Marie

    2012-01-01

    Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain. PMID:23185004

  13. 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

  14. Tyrosine-based signal mediates LRP6 receptor endocytosis and desensitization of Wnt/β-catenin pathway signaling.

    PubMed

    Liu, Chia-Chen; Kanekiyo, Takahisa; Roth, Barbara; Bu, Guojun

    2014-10-01

    Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases.

  15. Control of synaptic function by endocannabinoid-mediated retrograde signaling

    PubMed Central

    KANO, Masanobu

    2014-01-01

    Since the first reports in 2001, great advances have been made towards the understanding of endocannabinoid-mediated synaptic modulation. Electrophysiological studies have revealed that one of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG), is produced from membrane lipids upon postsynaptic Ca2+ elevation and/or activation of Gq/11-coupled receptors, and released from postsynaptic neurons. The released 2-AG then acts retrogradely onto presynaptic cannabinoid CB1 receptors and induces suppression of neurotransmitter release either transiently or persistently. These forms of 2-AG-mediated retrograde synaptic modulation are functional throughout the brain. The other major endocannabinoid, anandamide, mediates a certain form of endocannabinoid-mediated long-term depression (LTD). Anandamide also functions as an agonist for transient receptor potential vanilloid receptor type 1 (TRPV1) and mediates endocannabinoid-independent and TRPV1-dependent forms of LTD. It has also been demonstrated that the endocannabinoid system itself is plastic, which can be either up- or down-regulated by experimental or environmental conditions. In this review, I will make an overview of the mechanisms underlying endocannabinoid-mediated synaptic modulation. PMID:25169670

  16. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  17. The Notch signaling pathway as a mediator of tumor survival

    PubMed Central

    Pine, Sharon R.

    2013-01-01

    The Notch signaling pathway is evolutionarily conserved and responsible for cell fate determination in the developing embryo and mature tissue. At the molecular level, ligand binding activates Notch signaling by liberating the Notch intracellular domain, which then translocates into the nucleus and activates gene transcription. Despite the elegant simplicity of this pathway, which lacks secondary messengers or a signaling cascade, Notch regulates gene expression in a highly context- and cell-type-dependent manner. Notch signaling is frequently dysregulated, most commonly by overactivation, across many cancers and confers a survival advantage on tumors, leading to poorer outcomes for patients. Recent studies demonstrate how Notch signaling increases tumor cell proliferation and provide evidence that active Notch signaling maintains the cancer stem-cell pool, induces epithelial–mesenchymal transition and promotes chemoresistance. These studies imply that pharmacological inhibition of Notch signaling may refine control of cancer therapy and improve patient survival. Gamma secretase inhibitors (GSIs) are drugs that inhibit Notch signaling and may be successful in controlling cancer cell growth in conjunction with standard chemotherapy, but substantial side effects have hampered their widespread use. Recent efforts have been aimed at the development of antibodies against specific Notch receptors and ligands with the hope of limiting side effects while providing the same therapeutic benefit as GSIs. Together, studies characterizing Notch signaling and modulation have offered hope that refined methods targeting Notch may become powerful tools in anticancer therapeutics. PMID:23585460

  18. The Notch signaling pathway as a mediator of tumor survival.

    PubMed

    Capaccione, Kathleen M; Pine, Sharon R

    2013-07-01

    The Notch signaling pathway is evolutionarily conserved and responsible for cell fate determination in the developing embryo and mature tissue. At the molecular level, ligand binding activates Notch signaling by liberating the Notch intracellular domain, which then translocates into the nucleus and activates gene transcription. Despite the elegant simplicity of this pathway, which lacks secondary messengers or a signaling cascade, Notch regulates gene expression in a highly context- and cell-type-dependent manner. Notch signaling is frequently dysregulated, most commonly by overactivation, across many cancers and confers a survival advantage on tumors, leading to poorer outcomes for patients. Recent studies demonstrate how Notch signaling increases tumor cell proliferation and provide evidence that active Notch signaling maintains the cancer stem-cell pool, induces epithelial-mesenchymal transition and promotes chemoresistance. These studies imply that pharmacological inhibition of Notch signaling may refine control of cancer therapy and improve patient survival. Gamma secretase inhibitors (GSIs) are drugs that inhibit Notch signaling and may be successful in controlling cancer cell growth in conjunction with standard chemotherapy, but substantial side effects have hampered their widespread use. Recent efforts have been aimed at the development of antibodies against specific Notch receptors and ligands with the hope of limiting side effects while providing the same therapeutic benefit as GSIs. Together, studies characterizing Notch signaling and modulation have offered hope that refined methods targeting Notch may become powerful tools in anticancer therapeutics. PMID:23585460

  19. Calcium-Mediated Abiotic Stress Signaling in Roots.

    PubMed

    Wilkins, Katie A; Matthus, Elsa; Swarbreck, Stéphanie M; Davies, Julia M

    2016-01-01

    Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium's other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. PMID:27621742

  20. Calcium-Mediated Abiotic Stress Signaling in Roots

    PubMed Central

    Wilkins, Katie A.; Matthus, Elsa; Swarbreck, Stéphanie M.; Davies, Julia M.

    2016-01-01

    Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. PMID:27621742

  1. Calcium-Mediated Abiotic Stress Signaling in Roots

    PubMed Central

    Wilkins, Katie A.; Matthus, Elsa; Swarbreck, Stéphanie M.; Davies, Julia M.

    2016-01-01

    Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response.

  2. The Role of AGE/RAGE Signaling in Diabetes-Mediated Vascular Calcification

    PubMed Central

    2016-01-01

    AGE/RAGE signaling has been a well-studied cascade in many different disease states, particularly diabetes. Due to the complex nature of the receptor and multiple intersecting pathways, the AGE/RAGE signaling mechanism is still not well understood. The purpose of this review is to highlight key areas of AGE/RAGE mediated vascular calcification as a complication of diabetes. AGE/RAGE signaling heavily influences both cellular and systemic responses to increase bone matrix proteins through PKC, p38 MAPK, fetuin-A, TGF-β, NFκB, and ERK1/2 signaling pathways in both hyperglycemic and calcification conditions. AGE/RAGE signaling has been shown to increase oxidative stress to promote diabetes-mediated vascular calcification through activation of Nox-1 and decreased expression of SOD-1. AGE/RAGE signaling in diabetes-mediated vascular calcification was also attributed to increased oxidative stress resulting in the phenotypic switch of VSMCs to osteoblast-like cells in AGEs-induced calcification. Researchers found that pharmacological agents and certain antioxidants decreased the level of calcium deposition in AGEs-induced diabetes-mediated vascular calcification. By understanding the role the AGE/RAGE signaling cascade plays diabetes-mediated vascular calcification will allow for pharmacological intervention to decrease the severity of this diabetic complication. PMID:27547766

  3. The signaling symphony: T cell receptor tunes cytokine-mediated T cell differentiation.

    PubMed

    Huang, Weishan; August, Avery

    2015-03-01

    T cell development, differentiation, and maintenance are orchestrated by 2 key signaling axes: the antigen-specific TCR and cytokine-mediated signals. The TCR signals the recognition of self- and foreign antigens to control T cell homeostasis for immune tolerance and immunity, which is regulated by a variety of cytokines to determine T cell subset homeostasis and differentiation. TCR signaling can synergize with or antagonize cytokine-mediated signaling to fine tune T cell fate; however, the latter is less investigated. Murine models with attenuated TCR signaling strength have revealed that TCR signaling can function as regulatory feedback machinery for T cell homeostasis and differentiation in differential cytokine milieus, such as IL-2-mediated Treg development; IL-7-mediated, naïve CD8(+) T cell homeostasis; and IL-4-induced innate memory CD8(+) T cell development. In this review, we discuss the symphonic cross-talk between TCR and cytokine-mediated responses that differentially control T cell behavior, with a focus on the negative tuning by TCR activation on the cytokine effects.

  4. Tespa1 negatively regulates FcεRI-mediated signaling and the mast cell–mediated allergic response

    PubMed Central

    Zheng, Mingzhu; Qiu, Yuanjun; Guo, Chuansheng; Ji, Jian; Lei, Lei; Zhang, Xue; Liang, Jingjing; Lou, Jun; Huang, Wei; Dong, Bowen; Wu, Songquan; Wang, Jianli; Ke, Yuehai; Cao, Xuetao; Zhou, Yi Ting

    2014-01-01

    Antigen-mediated cross-linking of IgE on mast cells triggers a signaling cascade that results in their degranulation and proinflammatory cytokine production, which are key effectors in allergic reactions. We show that the activation of mast cells is negatively regulated by the newly identified adaptor protein Tespa1. Loss of Tespa1 in mouse mast cells led to hyper-responsiveness to stimulation via FcεRI. Mice lacking Tespa1 also displayed increased sensitivity to IgE-mediated allergic responses. The dysregulated signaling in KO mast cells was associated with increased activation of Grb2-PLC-γ1-SLP-76 signaling within the LAT1 (linker for activation of T cells family, member 1) signalosome versus the LAT2 signalosome. Collectively, these findings show that Tespa1 orchestrates mast cell activation by tuning the balance of LAT1 and LAT2 signalosome assembly. PMID:25422497

  5. Insulin Receptor Substrate 2 (IRS2)-Deficient Mice Show Sensorineural Hearing Loss That Is Delayed by Concomitant Protein Tyrosine Phosphatase 1B (PTP1B) Loss of Function

    PubMed Central

    Murillo-Cuesta, Silvia; Camarero, Guadalupe; González-Rodríguez, Águeda; de la Rosa, Lourdes Rodríguez; Burks, Deborah J; Avendaño, Carlos; Valverde, Ángela M; Varela-Nieto, Isabel

    2012-01-01

    The insulin receptor substrate (IRS) proteins are key mediators of insulin and insulinlike growth factor 1 (IGF-1) signaling. Protein tyrosine phosphatase (PTP)-1B dephosphorylates and inactivates both insulin and IGF-1 receptors. IRS2-deficient mice present altered hepatic insulin signaling and β-cell failure and develop type 2–like diabetes. In addition, IRS2 deficiency leads to developmental defects in the nervous system. IGF1 gene mutations cause syndromic sensorineural hearing loss in humans and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. Our objective was to study the hearing function and cochlear morphology of Irs2-null mice and the impact of PTP1B deficiency. We have studied the auditory brainstem responses and the cochlear morphology of systemic Irs2−/−Ptpn1+/+, Irs2+/+Ptpn1−/−and Irs2−/−Ptpn1−/− mice at different postnatal ages. The results indicated that Irs2−/−Ptpn1+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology with hypoinnervation of the cochlear ganglion and aberrant stria vascularis, compared with wild-type mice. Simultaneous PTP1B deficiency in Irs2−/−Ptpn1−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for treating deafness associated with hyperglycemia and type 2 diabetes. PMID:22160220

  6. Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons.

    PubMed

    Corey, Elizabeth A; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W

    2010-04-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a G protein-coupled receptor-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.

  7. An epidemic process mediated by a decaying diffusing signal

    NASA Astrophysics Data System (ADS)

    Faria, Fernando P.; Dickman, Ronald

    2012-06-01

    We study a stochastic epidemic model consisting of elements (organisms in a community or cells in tissue) with fixed positions, in which damage or disease is transmitted by diffusing agents ('signals') emitted by infected individuals. The signals decay as well as diffuse; since they are assumed to be produced in large numbers, the signal concentration is treated deterministically. The model, which includes four cellular states (susceptible, transformed, depleted, and removed), admits various interpretations: spread of an infection or infectious disease, or of damage in a tissue in which injured cells may themselves provoke further damage, and as a description of the so-called radiation-induced bystander effect, in which the signals are molecules capable of inducing cell damage and/or death in unirradiated cells. The model exhibits a continuous phase transition between spreading and nonspreading phases. We formulate two mean-field theory (MFT) descriptions of the model, one of which ignores correlations between the cellular state and the signal concentration, and another that treats such correlations in an approximate manner. Monte Carlo simulations of the spread of infection on the square lattice yield values for the critical exponents and the fractal dimension consistent with the dynamic percolation universality class.

  8. Chronic exposure to paclitaxel diminishes phosphoinositide signaling by calpain-mediated neuronal calcium sensor-1 degradation.

    PubMed

    Boehmerle, Wolfgang; Zhang, Kun; Sivula, Michael; Heidrich, Felix M; Lee, Yashang; Jordt, Sven-Eric; Ehrlich, Barbara E

    2007-06-26

    Paclitaxel (Taxol) is a well established chemotherapeutic agent for the treatment of solid tumors, but it is limited in its usefulness by the frequent induction of peripheral neuropathy. We found that prolonged exposure of a neuroblastoma cell line and primary rat dorsal root ganglia with therapeutic concentrations of Taxol leads to a reduction in inositol trisphosphate (InsP(3))-mediated Ca(2+) signaling. We also observed a Taxol-specific reduction in neuronal calcium sensor 1 (NCS-1) protein levels, a known modulator of InsP(3) receptor (InsP(3)R) activity. This reduction was also found in peripheral neuronal tissue from Taxol treated animals. We further observed that short hairpin RNA-mediated NCS-1 knockdown had a similar effect on phosphoinositide-mediated Ca(2+) signaling. When NCS-1 protein levels recovered, so did InsP(3)-mediated Ca(2+) signaling. Inhibition of the Ca(2+)-activated protease mu-calpain prevented alterations in phosphoinositide-mediated Ca(2+) signaling and NCS-1 protein levels. We also found that NCS-1 is readily degraded by mu-calpain in vitro and that mu-calpain activity is increased in Taxol but not vehicle-treated cells. From these results, we conclude that prolonged exposure to Taxol activates mu-calpain, which leads to the degradation of NCS-1, which, in turn, attenuates InsP(3)mediated Ca(2+) signaling. These findings provide a previously undescribed approach to understanding and treating Taxol-induced peripheral neuropathy. PMID:17581879

  9. Notch signalling mediates reproductive constraint in the adult worker honeybee

    PubMed Central

    Duncan, Elizabeth J.; Hyink, Otto; Dearden, Peter K.

    2016-01-01

    The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen. PMID:27485026

  10. Notch signalling mediates reproductive constraint in the adult worker honeybee.

    PubMed

    Duncan, Elizabeth J; Hyink, Otto; Dearden, Peter K

    2016-01-01

    The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen. PMID:27485026

  11. Discovering mechanisms of signaling-mediated cysteine oxidation.

    PubMed

    Poole, Leslie B; Nelson, Kimberly J

    2008-02-01

    Accumulating evidence reveals hydrogen peroxide as a key player both as a damaging agent and, from emerging evidence over the past decade, as a second messenger in intracellular signaling. This rather mild oxidant acts upon downstream targets within signaling cascades to modulate the activity of a host of enzymes (e.g. phosphatases and kinases) and transcriptional regulators through chemoselective oxidation of cysteine residues. With the recent development of specific detection reagents for hydrogen peroxide and new chemical tools to detect the generation of the initial oxidation product, sulfenic acid, on reactive cysteines within target proteins, the scene is set to gain a better understanding of the mechanisms through which hydrogen peroxide acts as a second messenger in cell signaling.

  12. Role of Reactive Oxygen Species-Mediated Signaling in Aging

    PubMed Central

    Labunskyy, Vyacheslav M.

    2013-01-01

    Abstract Significance: Redox biology is a rapidly developing area of research due to the recent evidence for general importance of redox control for numerous cellular functions under both physiological and pathophysiological conditions. Understanding of redox homeostasis is particularly relevant to the understanding of the aging process. The link between reactive oxygen species (ROS) and accumulation of age-associated oxidative damage to macromolecules is well established, but remains controversial and applies only to a subset of experimental models. In addition, recent studies show that ROS may function as signaling molecules and that dysregulation of this process may also be linked to aging. Recent Advances: Many protein factors and pathways that control ROS production and scavenging, as well as those that regulate cellular redox homeostasis, have been identified. However, much less is known about the mechanisms by which redox signaling pathways influence longevity. In this review, we discuss recent advances in the understanding of the molecular basis for the role of redox signaling in aging. Critical Issues: Recent studies allowed identification of previously uncharacterized redox components and revealed complexity of redox signaling pathways. It would be important to identify functions of these components and elucidate how distinct redox pathways are integrated with each other to maintain homeostatic balance. Future Directions: Further characterization of processes that coordinate redox signaling, redox homeostasis, and stress response pathways should allow researchers to dissect how their dysregulation contributes to aging and pathogenesis of various age-related diseases, such as diabetes, cancer and neurodegeneration. Antioxid. Redox Signal. 19, 1362–1372. PMID:22901002

  13. Specificity is complex and time consuming: mutual exclusivity in tyrosine kinase-mediated signaling.

    PubMed

    O'Rourke, Lisa; Ladbury, John E

    2003-06-01

    Most fundamental cellular processes are transduced through tyrosine kinase (TK)-mediated pathways. For transduction without corruption, the protein-protein interactions involved have to be mutually exclusive. Many of these proteins bind via homologous domains whose binding characteristics suggest that their innate specificity is not sufficiently high to account for the integrity of signal transduction. Stimulation of TK-mediated signals is often accompanied by recruitment of a precise, multimolecular protein complex that is itself capable of imposing specificity. Furthermore, this complex provides protection against phosphatase activity, controlling the longevity of the active signaling complex, and thus influencing outcomes in subsequent downstream events.

  14. Statil suppresses cancer cell growth and proliferation by the inhibition of tumor marker AKR1B10.

    PubMed

    Cao, Zhe; Zhou, Boping; Chen, Xinchun; Huang, Dan; Zhang, Xiuli; Wang, Ziqi; Huang, Hua; Wang, Yuhong; Cao, Deliang

    2014-09-01

    Aldo-keto reductase 1B10 (AKR1B10) is an oncogenic carbonyl reductase that eliminates α,β-unsaturated carbonyl compounds/lipid peroxides and mediates retinoic acid signaling. Targeted inhibition of AKR1B10 activity is a newly emerging strategy for cancer therapy. This study evaluated the inhibitory activity of a small chemical statil towards AKR1B10 and tested its antiproliferative activity in breast (BT-20) and lung (NCI-H460) cancer cells that express AKR1B10. Experimental results showed that statil inhibited AKR1B10 enzyme activity efficiently, with an IC50 at 0.21±0.06 µmol/l. Exposing BT-20 and NCI-H460 cells to statil and diclofenac, a selective AKR1B10 inhibitor, led to dose-dependent inhibition of cell growth and proliferation and plating efficiency. At higher doses (50 µmol/l or higher), statil induced cell death with apoptotic characteristics, such as DNA fragmentation and Annexin-V staining. Furthermore, statil enhanced the susceptibility of cells to acrolein, an active substrate of AKR1B10. Taken together, these data suggest that statil possesses potent antiproliferative activity by inhibiting AKR1B10 activity.

  15. Octadecanoid-Mediated Signal Transduction in Higher Plants

    NASA Astrophysics Data System (ADS)

    Weiler, Elmar W.

    The observation that methyljasmonate is a strong promoter of senescence marked the discovery of lipid-derived signaling molecules of higher plants. This group of compounds, now collectively termed octadecanoids, is derived from the fatty acid α-linolenic acid and involved in physiological processes such diverse as the triggering of defense reactions against herbivores and pathogens, mechanotransduction, plant volatile emission, potato tuberization, and many others. Recent research has yielded clues to a deeper understanding of octadecanoid biology. Control over this central signaling system may open new avenues in biological pest control through plant defense regulators.

  16. Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity.

    PubMed

    Zeaiter, Zaher; Cohen, David; Müsch, Anne; Bagnoli, Fabio; Covacci, Antonello; Stein, Markus

    2008-03-01

    Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation. PMID:18005242

  17. Calcium signaling as a mediator of cell energy demand and a trigger to cell death.

    PubMed

    Bhosale, Gauri; Sharpe, Jenny A; Sundier, Stephanie Y; Duchen, Michael R

    2015-09-01

    Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury.

  18. Calcium signaling as a mediator of cell energy demand and a trigger to cell death

    PubMed Central

    Bhosale, Gauri; Sharpe, Jenny A.; Sundier, Stephanie Y.

    2015-01-01

    Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury. PMID:26375864

  19. The canonical Wg signaling modulates Bsk-mediated cell death in Drosophila

    PubMed Central

    Zhang, S; Chen, C; Wu, C; Yang, Y; Li, W; Xue, L

    2015-01-01

    Cell death is an essential regulatory mechanism for removing unneeded cells in animal development and tissue homeostasis. The c-Jun N-terminal kinase (JNK) pathway has pivotal roles in the regulation of cell death in response to various intrinsic and extrinsic stress signals. The canonical Wingless (Wg) signaling has been implicated in cell proliferation and cell fate decisions, whereas its role in cell death remains largely elusive. Here, we report that activated Bsk (the Drosophila JNK homolog) induced cell death is mediated by the canonical Wg signaling. First, loss of Wg signaling abrogates Bsk-mediated caspase-independent cell death. Second, activation of Wg signaling promotes cell death in a caspase-independent manner. Third, activation of Bsk signaling results in upregulated transcription of wingless (wg) gene. Finally, Wg pathway participates in the physiological function of Bsk signaling in development. These findings not only reveal a previously undiscovered role of Wg signaling in Bsk-mediated cell death, but also provide a novel mechanism for the interplay between the two important signaling pathways in development. PMID:25855961

  20. AIP1-mediated stress signaling in atherosclerosis and arteriosclerosis.

    PubMed

    Zhang, Jiqin; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

    2015-05-01

    AIP1 (ASK1-interacting protein-1; encoded by the DAB2IP gene), a signaling scaffolding protein, is abundantly expressed in vascular endothelial cells (EC). While it was initially discovered as an apoptosis signal-regulating kinase 1 (ASK1)-interacting protein, AIP1 broadly suppresses inflammatory responses triggered by cytokines and stresses such as TNF, LPS, VEGF, and endoplasmic reticulum (ER) stress in EC (therefore, AIP1 is an anti-inflammatory protein). Human genome-wide association study (GWAS) has identified DAB2IP gene variants conferring susceptibility to cardiovascular diseases. Consistently, a global or vascular EC-specific deletion of DAB2IP in mice strongly enhances inflammatory responses and exacerbates atherosclerosis and graft arteriosclerosis progression in mouse models. Mechanisms for AIP1 function and regulation associated with human cardiovascular diseases need further investigations.

  1. New Insights into Reelin-Mediated Signaling Pathways

    PubMed Central

    Lee, Gum Hwa; D’Arcangelo, Gabriella

    2016-01-01

    Reelin, a multifunctional extracellular protein that is important for mammalian brain development and function, is secreted by different cell types in the prenatal or postnatal brain. The spatiotemporal regulation of Reelin expression and distribution during development relates to its multifaceted function in the brain. Prenatally Reelin controls neuronal radial migration and proper positioning in cortical layers, whereas postnatally Reelin promotes neuronal maturation, synaptic formation and plasticity. The molecular mechanisms underlying the distinct biological functions of Reelin during and after brain development involve unique and overlapping signaling pathways that are activated following Reelin binding to its cell surface receptors. Distinct Reelin ligand isoforms, such as the full-length protein or fragments generated by proteolytic cleavage differentially affect the activity of downstream signaling pathways. In this review, we discuss recent advances in our understanding of the signaling transduction pathways activated by Reelin that regulate different aspects of brain development and function. A core signaling machinery, including ApoER2/VLDLR receptors, Src/Fyn kinases, and the adaptor protein Dab1, participates in all known aspects of Reelin biology. However, distinct downstream mechanisms, such as the Crk/Rap1 pathway and cell adhesion molecules, play crucial roles in the control of neuronal migration, whereas the PI3K/Akt/mTOR pathway appears to be more important for dendrite and spine development. Finally, the NMDA receptor (NMDAR) and an unidentified receptor contribute to the activation of the MEK/Erk1/2 pathway leading to the upregulation of genes involved in synaptic plasticity and learning. This knowledge may provide new insight into neurodevelopmental or neurodegenerative disorders that are associated with Reelin dysfunction. PMID:27242434

  2. Structural basis for angiopoietin-1–mediated signaling initiation

    SciTech Connect

    Yu, Xuehong; Seegar, Tom C. M.; Dalton, Annamarie C.; Tzvetkova-Robev, Dorothea; Goldgur, Yehuda; Rajashankar, Kanagalaghatta R.; Nikolov, Dimitar B.; Barton, William A.

    2013-04-30

    Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

  3. Specific inhibition of sensitized protein tyrosine phosphatase 1B (PTP1B) with a biarsenical probe

    PubMed Central

    Davis, Oliver B.; Bishop, Anthony C.

    2012-01-01

    Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of the insulin-receptor and leptin-receptor signaling pathways, and it has therefore emerged as a critical anti-type-II-diabetes and anti-obesity drug target. Toward the goal of generating a covalent modulator of PTP1B activity that can be used for investigating its roles in cell signaling and disease progression, we report that the biarsenical probe FlAsH-EDT2 can be used to inhibit PTP1B variants that contain cysteine point mutations in a key catalytic loop of the enzyme. The site-specific cysteine mutations have little effect on the catalytic activity of the enzyme in the absence of FlAsH-EDT2. Upon addition of FlAsH-EDT2, however, the activity of the engineered PTP1B is strongly inhibited, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that the cysteine-rich PTP1B variants can be targeted with the biarsenical probe in either whole-cell lysates or intact cells. Together, our data provide an example of a biarsenical probe controlling the activity of a protein that does not contain the canonical tetra-cysteine biarsenical-labeling sequence CCXXCC. The targeting of “incomplete” cysteine-rich motifs could provide a general means for controlling protein activity by targeting biarsenical compounds to catalytically important loops in conserved protein domains. PMID:22263876

  4. PPARs Mediate Lipid Signaling in Inflammation and Cancer

    PubMed Central

    Michalik, Liliane; Wahli, Walter

    2008-01-01

    Lipid mediators can trigger physiological responses by activating nuclear hormone receptors, such as the peroxisome proliferator-activated receptors (PPARs). PPARs, in turn, control the expression of networks of genes encoding proteins involved in all aspects of lipid metabolism. In addition, PPARs are tumor growth modifiers, via the regulation of cancer cell apoptosis, proliferation, and differentiation, and through their action on the tumor cell environment, namely, angiogenesis, inflammation, and immune cell functions. Epidemiological studies have established that tumor progression may be exacerbated by chronic inflammation. Here, we describe the production of the lipids that act as activators of PPARs, and we review the roles of these receptors in inflammation and cancer. Finally, we consider emerging strategies for therapeutic intervention. PMID:19125181

  5. [The interactions between natural products and OATP1B1].

    PubMed

    Shi, Mei-zhi; Liu, Yu; Bian, Jia-lin; Jin, Meng; Gui, Chun-shan

    2015-07-01

    Organic anion transporting polypeptide 1B1 (OATP1B1) is an important liver-specific uptake transporter, which mediates transport of numerous endogenous substances and drugs from blood into hepatocytes. To identify and investigate potential modulators of OATP1B1 from natural products, the effect of 21 frequently used natural compounds and extracts on OATP1B1-mediated fluorescein methotrexate transport was studied by using Chinese hamster ovary cells stably expressing OATP1B1 (CHO-OATP1B1) in 96-well plates. This method could be used for the screening of large compound libraries. Our studies showed that some flavonoids (e.g., quercetin, quercitrin, rutin, chrysanthemum flavonoids and mulberrin) and triterpenoids (e.g., glycyrrhetinic acid and glycyrrhizic acid) were inhibitors of OATP1B1 with IC50 values less than 16 µmol · L(-1). The IC50 value of glycyrrhetinic acid on OATP1B1 was comparable to its blood concentration in clinics, indicating an OATPlB1-mediated drug-drug interaction could occur. Structure-activity relationship analysis showed that flavonoids had much higher inhibitory activity than their glycosides. Furthermore, the type and length of saccharides had a significant effect on their activity. In addition, we used OATP1B1 substrates fluvastatin and rosuvastatin as probe drugs to investigate the substrate-dependent effect of several natural compounds on the function of OATP1B1 in vitro. Our results demonstrated that the effect of these natural products on the function of OATPlB1 was substrate-dependent. In summary, this study would be conducive to predicting and avoiding potential OATP1B1-mediated drug-drug and drug-food interactions and thus provide the experimental basis and guidance for rational drug use. PMID:26552146

  6. Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum.

    PubMed

    Willard, Stacey S; Devreotes, Peter N

    2006-09-01

    Chemotaxis, or cell migration guided by chemical cues, is critical for a multitude of biological processes in a diverse array of organisms. Dictyostelium discoideum amoebae rely on chemotaxis to find food and to survive starvation conditions, and we have taken advantage of this system to study the molecular regulation of this vital cell behavior. Previous work has identified phosphoinositide signaling as one mechanism which may contribute to directional sensing and actin polymerization during chemotaxis; a mechanism which is conserved in mammalian neutrophils. In this review, we will discuss recent data on genes and pathways governing directional sensing and actin polymerization, with a particular emphasis on contributions from our laboratory. PMID:16962888

  7. The polycystin complex mediates Wnt/Ca(2+) signalling.

    PubMed

    Kim, Seokho; Nie, Hongguang; Nesin, Vasyl; Tran, Uyen; Outeda, Patricia; Bai, Chang-Xi; Keeling, Jacob; Maskey, Dipak; Watnick, Terry; Wessely, Oliver; Tsiokas, Leonidas

    2016-07-01

    WNT ligands induce Ca(2+) signalling on target cells. PKD1 (polycystin 1) is considered an orphan, atypical G-protein-coupled receptor complexed with TRPP2 (polycystin 2 or PKD2), a Ca(2+)-permeable ion channel. Inactivating mutations in their genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases. Here, we show that WNTs bind to the extracellular domain of PKD1 and induce whole-cell currents and Ca(2+) influx dependent on TRPP2. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or reduce TRPP2 channel activity suppress activation by WNTs. Pkd2(-/-) fibroblasts lack WNT-induced Ca(2+) currents and are unable to polarize during directed cell migration. In Xenopus embryos, pkd1, Dishevelled 2 (dvl2) and wnt9a act within the same pathway to preserve normal tubulogenesis. These data define PKD1 as a WNT (co)receptor and implicate defective WNT/Ca(2+) signalling as one of the causes of ADPKD. PMID:27214281

  8. Hypospadias and anorectal malformations mediated by Eph/ephrin signaling

    PubMed Central

    Yucel, Selcuk; Dravis, Christopher; Garcia, Nilda; Henkemeyer, Mark; Baker, Linda A.

    2007-01-01

    Purpose Despite extensive research, the molecular basis of hypospadias and anorectal malformations is poorly understood, likely due to a multifactorial basis. The incidence of hypospadias is increasing, thus making research in this area warranted and timely. This review presents recent molecular work broadening our understanding of these disorders. Materials and Methods A brief review of our recent work and the literature on the role of Eph/ephrin signaling in hypospadias and anorectal malformations is presented. Results Genetically engineered mice mutant for ephrin-B2 or EphB2;EphB3 manifest a variety of genitourinary and anorectal malformations. Approximately 40% of adult male heterozygous mice demonstrate perineal hypospadias. Although homozygous mice die soon after birth, 100% of homozygous males demonstrate high imperforate anus with urethral anomalies and 100% of homozygous females demonstrate persistent cloaca. Male mice compound homozygous for EphB2ki/ki;EphB3Δ/Δ/ also demonstrate hypospadias. Conclusions These mouse models provide compelling evidence of the role of B-class Eph/ephrin signaling in genitourinary/anorectal development and add to our mechanistic and molecular understanding of normal and abnormal embryonic development. As research on the B-class Ephs and ephrins continues, they will likely be shown to be molecular contributors to the multifactorial basis of hypospadias and anorectal malformations in humans as well. PMID:18431460

  9. Interferon Beta-1b Injection

    MedlinePlus

    ... course of disease where symptoms flare up from time to time) of multiple sclerosis (MS, a disease in which ... interferon beta-1b injection at around the same time of day each time you inject it. Follow ...

  10. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

    PubMed Central

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E.; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-01-01

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits. PMID:25916845

  11. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

    PubMed

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-05-26

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

  12. Signals mediating Klotho-induced neuroprotection in hippocampal neuronal cells.

    PubMed

    Cheng, Meng-Fu; Chen, Li-Jen; Niu, Ho-Shan; Yang, Ting-Ting; Lin, Kao-Chang; Cheng, Juei-Tang

    2015-01-01

    The erythropoietin (Epo) receptor (EpoR) is expressed in the brain and was shown to have neuroprotective effects against brain damage in animal models. A recent study indicated that EpoR and its activity are the downstream effectors of Klotho for cytoprotection in the kidney. Thus, we propose that Klotho can stimulate the expression of EpoR in neuronal cells to enhance Epo-mediated protection. H19-7 hippocampal neuronal cells were treated with recombinant Klotho. In H19-7 cells, Klotho increased the expression of both the EpoR protein and mRNA. Klotho also enhanced the transcription activity of the EpoR promoter in H19-7 cells. Moreover, Klotho augmented the Epo-triggered phosphorylation of Jak2 and Stat5 and protected H19-7 cells from hydrogen peroxide cytotoxicity. The silencing of EpoR abolished the protective effect of Klotho against peroxide-induced cytotoxicity. Finally, the silencing of GATA1 diminished the Klotho-induced increase in EpoR protein and mRNA expression as well as its promoter activity. In conclusion, Klotho increased EpoR expression in neuronal cells through GATA1, thereby enabling EpoR to function as a cytoprotective protein against oxidative injury. PMID:25856523

  13. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

    PubMed

    Irani, K; Xia, Y; Zweier, J L; Sollott, S J; Der, C J; Fearon, E R; Sundaresan, M; Finkel, T; Goldschmidt-Clermont, P J

    1997-03-14

    NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

  14. Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

    PubMed Central

    Sargoy, Allison; Sun, Xiaoping

    2014-01-01

    Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury. PMID:24416240

  15. AIP1-mediated stress signaling in atherosclerosis and arteriosclerosis

    PubMed Central

    Zhang, Jiqin; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

    2016-01-01

    AIP1 (encoded by the DAB2IP gene), a signaling scaffolding protein, is abundantly expressed in vascular endothelial cells (EC). While it was initially discovered as an ASK1-interacting protein, AIP1 broadly suppresses inflammatory responses triggered by cytokines and stresses such as TNF, LPS, VEGF and ER stress in EC (therefore AIP1 is an Anti-Inflammatory Protein). Human genome-wide association study (GWAS) has identified DAB2IP gene variants conferring susceptibility to cardiovascular diseases. Consistently, a global or vascular EC-specific deletion of DAB2IP in mice strongly enhances inflammatory responses and exacerbates atherosclerosis and graft arteriosclerosis progression in mouse models. Mechanisms for AIP1 function and regulation associated with human cardiovascular diseases need further investigations. PMID:25732743

  16. Illuminating Progress in Phytochrome-Mediated Light Signaling Pathways.

    PubMed

    Xu, Xiaosa; Paik, Inyup; Zhu, Ling; Huq, Enamul

    2015-10-01

    Light signals regulate a plethora of plant responses throughout their life cycle, especially the red and far-red regions of the light spectrum perceived by the phytochrome family of photoreceptors. However, the mechanisms by which phytochromes regulate gene expression and downstream responses remain elusive. Several recent studies have unraveled the details on how phytochromes regulate photomorphogenesis. These include the identification of E3 ligases that degrade PHYTOCHROME INTERACTING FACTOR (PIF) proteins, key negative regulators, in response to light, a better view of how phytochromes inhibit another key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and an understanding of why plants evolved multiple negative regulators to repress photomorphogenesis in darkness. These advances will surely fuel future research on many unanswered questions that have intrigued plant photobiologists for decades.

  17. Staphylococcal Superantigens Spark Host-Mediated Danger Signals

    PubMed Central

    Krakauer, Teresa; Pradhan, Kisha; Stiles, Bradley G.

    2016-01-01

    Staphylococcal enterotoxin B (SEB) of Staphylococcus aureus, and related superantigenic toxins produced by myriad microbes, are potent stimulators of the immune system causing a variety of human diseases from transient food poisoning to lethal toxic shock. These protein toxins bind directly to specific Vβ regions of T-cell receptors (TCR) and major histocompatibility complex (MHC) class II on antigen-presenting cells, resulting in hyperactivation of T lymphocytes and monocytes/macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. Because of superantigen-induced T cells skewed toward TH1 helper cells, and the induction of proinflammatory cytokines, superantigens can exacerbate autoimmune diseases. Upon TCR/MHC ligation, pathways induced by superantigens include the mitogen-activated protein kinase cascades and cytokine receptor signaling, resulting in activation of NFκB and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. Various mouse models exist to study SEB-induced shock including those with potentiating agents, transgenic mice and an “SEB-only” model. However, therapeutics to treat toxic shock remain elusive as host response genes central to pathogenesis of superantigens have only been identified recently. Gene profiling of a murine model for SEB-induced shock reveals novel molecules upregulated in multiple organs not previously associated with SEB-induced responses. The pivotal genes include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, immunoproteasome components, as well as antiviral and IFN-stimulated genes. The host-wide induction of these, and other, antimicrobial defense genes provide evidence that SEB elicits danger signals resulting in multi-organ damage and toxic

  18. Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells.

    PubMed

    Sosna, Justyna; Philipp, Stephan; Fuchslocher Chico, Johaiber; Saggau, Carina; Fritsch, Jürgen; Föll, Alexandra; Plenge, Johannes; Arenz, Christoph; Pinkert, Thomas; Kalthoff, Holger; Trauzold, Anna; Schmitz, Ingo; Schütze, Stefan; Adam, Dieter

    2016-10-15

    Recently, a type of regulated necrosis (RN) called necroptosis was identified to be involved in many pathophysiological processes and emerged as an alternative method to eliminate cancer cells. However, only a few studies have elucidated components of TRAIL-mediated necroptosis useful for anticancer therapy. Therefore, we have compared this type of cell death to tumor necrosis factor (TNF)-mediated necroptosis and found similar signaling through acid and neutral sphingomyelinases, the mitochondrial serine protease HtrA2/Omi, Atg5, and vacuolar H(+)-ATPase. Notably, executive mechanisms of both TRAIL- and TNF-mediated necroptosis are independent of poly(ADP-ribose) polymerase 1 (PARP-1), and depletion of p38α increases the levels of both types of cell death. Moreover, we found differences in signaling between TNF- and TRAIL-mediated necroptosis, e.g., a lack of involvement of ubiquitin carboxyl hydrolase L1 (UCH-L1) and Atg16L1 in executive mechanisms of TRAIL-mediated necroptosis. Furthermore, we discovered indications of an altered involvement of mitochondrial components, since overexpression of the mitochondrial protein Bcl-2 protected Jurkat cells from TRAIL- and TNF-mediated necroptosis, and overexpression of Bcl-XL diminished only TRAIL-induced necroptosis in Colo357 cells. Furthermore, TRAIL does not require receptor internalization and endosome-lysosome acidification to mediate necroptosis. Taken together, pathways described for TRAIL-mediated necroptosis and differences from those for TNF-mediated necroptosis might be unique targets to increase or modify necroptotic signaling and eliminate tumor cells more specifically in future anticancer approaches. PMID:27528614

  19. PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects.

    PubMed

    Huang, Jianhua; Kontos, Christopher D

    2002-03-29

    Phosphatidylinositol 3-kinase is activated by vascular endothelial growth factor (VEGF), and many of the angiogenic cellular responses of VEGF are regulated by the lipid products of phosphatidylinositol 3-kinase. The tumor suppressor PTEN has been shown to down-regulate phosphatidylinositol 3-kinase signaling, yet the effects of PTEN on VEGF-mediated signaling and angiogenesis are unknown. Inhibition of endogenous PTEN in cultured endothelial cells by adenovirus-mediated overexpression of a dominant negative PTEN mutant (PTEN-C/S) enhanced VEGF-mediated Akt phosphorylation, and this effect correlated with decreases in caspase-3 cleavage, caspase-3 activity, and DNA degradation after induction of apoptosis with tumor necrosis factor-alpha. Overexpression of PTEN-C/S also enhanced VEGF-mediated endothelial cell proliferation and migration. In contrast, overexpression of wild-type PTEN inhibited the anti-apoptotic, proliferative, and chemotactic effects of VEGF. Moreover, PTEN-C/S increased the length of vascular sprouts in the rat aortic ring assay and modulated VEGF-mediated tube formation in an in vitro angiogenesis assay, whereas PTEN-wild type inhibited these effects. Taken together, these findings demonstrate that PTEN potently modulates VEGF-mediated signaling and function and that PTEN is a viable target in therapeutic approaches to promote or inhibit angiogenesis.

  20. HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.

    PubMed

    Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

    2012-01-01

    HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.

  1. Arabinosylated lipoarabinomannan (Ara-LAM) mediated intracellular mechanisms against tuberculosis infection: involvement of protein kinase C (PKC) mediated signaling.

    PubMed

    Das, Shibali; Bhattacharjee, Oindrila; Goswami, Avranil; Pal, Nishith K; Majumdar, Subrata

    2015-03-01

    Tuberculosis causes severe immunosuppression thereby ensuring the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates TLR-2 receptor down-stream signaling, indicating the possible involvement of TLR-2 in the regulation of the host immune response. Moreover, different PKC isoforms are also involved in the course of infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immuno-modulatory properties which induce the pro-inflammatory responses via induction of TLR-2-mediated signaling. Here, we found that pretreatment of M. tuberculosis-infected macrophages with Ara-LAM caused a significant increase in the conventional PKC expression along with their active association with TLR-2. This association activated the TLR-2 -mediated downstream signaling, facilitating the activation of MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response, which was abrogated by treatment with PKC-α and P38 inhibitors. Moreover, pretreatment of macrophages with Ara-LAM abrogated the IL-10 production while restored MHC-II expression in the infected macrophages. This study demonstrates that Ara-LAM confers protection against tuberculosis via TLR-2/PKC signaling crosstalk which is responsible for the induction of host protective immune response against tuberculosis.

  2. Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish.

    PubMed

    Lovely, C Ben; Swartz, Mary E; McCarthy, Neil; Norrie, Jacqueline L; Eberhart, Johann K

    2016-06-01

    The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. PMID:27122171

  3. Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

    PubMed

    Roy, Nivedita; Chakraborty, Supriya; Paul Chowdhury, Bidisha; Banerjee, Sayantan; Halder, Kuntal; Majumder, Saikat; Majumdar, Subrata; Sen, Parimal C

    2014-01-01

    Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis. PMID:25329062

  4. TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin

    PubMed Central

    Yabe-Wada, Toshiki; Matsuba, Shintaro; Takeda, Kazuya; Sato, Tetsuya; Suyama, Mikita; Ohkawa, Yasuyuki; Takai, Toshiyuki; Shi, Haifeng; Philpott, Caroline C.; Nakamura, Akira

    2016-01-01

    Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3′ UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals. PMID:27220277

  5. Plasmodesmata-mediated intercellular signaling during plant growth and development

    PubMed Central

    Yadav, Shri R.; Yan, Dawei; Sevilem, Iris; Helariutta, Ykä

    2014-01-01

    Plasmodesmata (PD) are cytoplasmic channels that connect neighboring cells for cell-to-cell communication. PD structure and function vary temporally and spatially to allow formation of symplastic domains during different stages of plant development. Reversible deposition of callose at PD plays an important role in controlling molecular trafficking through PD by regulating their size exclusion limit. Previously, we reported several semi-dominant mutants for CALLOSE SYNTHASE 3 (CALS3) gene, which overproduce callose at PD in Arabidopsis. By combining two of these mutations in a LexA-VP16-ER (XVE)-based estradiol inducible vector system, a tool known as the “icals3m system” was developed to temporally obstruct the symplastic connections in a specified spatial domain. The system has been successfully tested and used, in combination with other methods, to investigate the route for mobile signals such as the SHR protein, microRNA165/6, and cytokinins in Arabidopsis roots, and also to understand the role of symplastic domain formation during lateral root development. We envision that this tool may also be useful for identifying tissue-specific symplastic regulatory networks and to analyze symplastic movement of metabolites. PMID:24596574

  6. Performing a hepatic timing signal: glucocorticoids induce gper1a and gper1b expression and repress gclock1a and gbmal1a in the liver of goldfish.

    PubMed

    Sánchez-Bretaño, Aída; Callejo, María; Montero, Marta; Alonso-Gómez, Ángel L; Delgado, María J; Isorna, Esther

    2016-01-01

    Glucocorticoids have been recently proposed as input signals of circadian system, although the underlying molecular mechanism remains unclear. This work investigates the role of glucocorticoids as modulators of clock genes expression in the liver of goldfish. In fish maintained under a 12L:12D photoperiod, an intraperitoneal injection at Zeitgeber Time 2 of a glucocorticoid analog, dexamethasone (1 μg/g body weight) induced per1 genes while decreased gbmal1a and gclock1a expression in the liver at 8 h post-injection. A 4-h in vitro exposure of goldfish liver to cortisol (0.1-10 μM) also induced gper1 genes in a concentration-dependent manner. Similarly, the exposure of the goldfish cultured liver to dexamethasone produced a concentration-dependent induction of gper1 genes. Moreover, this glucocorticoid analog led to a decrease in gbmal1a and gclock1a transcripts, while the other clock genes analyzed were unaffected. The induction of gper1a and gper1b by dexamethasone in vitro was observed at short times (2 h), whereas the reductions of gbmal1a and gclock1a transcripts needed longer exposure times (8 h) to the glucocorticoid to be significant. Additionally, a 2-h exposure to dexamethasone in the liver culture was enough to extend the induction of per genes for more than 12 h. Present results indicate that gper1 genes are targets for glucocorticoids in the regulation of goldfish hepatic oscillator, as previously reported in mammals, suggesting a conserved role of glucocorticoids in the functional organization of the peripheral circadian system in vertebrates. The repression of clock1a and bmal1a is not so well established, and suggests that other clock genes could be glucocorticoid targets in the goldfish liver.

  7. Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to post-fracture nociceptive sensitization

    PubMed Central

    Shi, Xiaoyou; Guo, Tian-zhi; Wei, Tzuping; Li, Wen-wu; Clark, David J; Kingery, Wade S

    2015-01-01

    Tibia fracture induces exaggerated substance P (SP) and CGRP signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hindlimbs of rats similar to those seen in complex regional pain syndrome (CRPS). Inflammatory changes in the spinal cord contribute to nociceptive sensitization in a variety of animal pain models. This study tested the hypothesis that fracture induced exaggerated neuropeptide signaling up-regulates spinal inflammatory mediator expression, leading to post-fracture hindlimb nociceptive sensitization. At 4 weeks after performing tibia fracture and casting in rats, we measured hindlimb allodynia, unweighting, warmth, edema, and spinal cord neuropeptide and inflammatory mediator content. The antinociceptive effects of intrathecally injected neuropeptide and inflammatory mediator receptor antagonists were evaluated in fracture rats. Transgenic fracture mice lacking SP or the CGRP RAMP1 receptor were used to determine the effects of neuropeptide signaling on post-fracture pain behavior and spinal inflammatory mediator expression. Hindlimb allodynia, unweighting, warmth, edema, increased spinal SP and CGRP, and increased spinal inflammatory mediator expression (TNF, IL-1, IL-6, CCL2, NGF) were observed at 4 weeks after fracture in rats. Fracture induced increases in spinal inflammatory mediators were not observed in fracture mice lacking SP or the CGRP receptor and these mice had attenuated post-fracture nociceptive sensitization. Intrathecal injection of selective receptor antagonists for SP, CGRP, TNF, IL-1, IL-6, CCL2, or NGF each reduced pain behaviors in the fracture rats. Collectively, these data support the hypothesis that facilitated spinal neuropeptide signaling up-regulates the expression of spinal inflammatory mediators contributing to nociceptive sensitization in a rodent fracture model of CRPS. PMID:25932690

  8. Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to postfracture nociceptive sensitization.

    PubMed

    Shi, Xiaoyou; Guo, Tian-Zhi; Wei, Tzuping; Li, Wen-Wu; Clark, David J; Kingery, Wade S

    2015-10-01

    Tibia fracture induces exaggerated substance P (SP) and calcitonin gene-related peptide (CGRP) signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hind limbs of rats similar to those seen in complex regional pain syndrome. Inflammatory changes in the spinal cord contribute to nociceptive sensitization in a variety of animal pain models. This study tested the hypothesis that fracture-induced exaggerated neuropeptide signaling upregulates spinal inflammatory mediator expression, leading to postfracture hind limb nociceptive sensitization. At 4 weeks after performing tibia fracture and casting in rats, we measured hind limb allodynia, unweighting, warmth, edema, and spinal cord neuropeptide and inflammatory mediator content. The antinociceptive effects of intrathecally injected neuropeptide and inflammatory mediator receptor antagonists were evaluated in fracture rats. Transgenic fracture mice lacking SP or the CGRP RAMP1 receptor were used to determine the effects of neuropeptide signaling on postfracture pain behavior and spinal inflammatory mediator expression. Hind limb allodynia, unweighting, warmth, edema, increased spinal SP and CGRP, and increased spinal inflammatory mediator expression (TNF, IL-1, IL-6, CCL2, and nerve growth factor) were observed at 4 weeks after fracture in rats. Fracture-induced increases in spinal inflammatory mediators were not observed in fracture mice lacking SP or the CGRP receptor, and these mice had attenuated postfracture nociceptive sensitization. Intrathecal injection of selective receptor antagonists for SP, CGRP, TNF, IL-1, IL-6, CCL2, or nerve growth factor each reduced pain behaviors in the fracture rats. Collectively, these data support the hypothesis that facilitated spinal neuropeptide signaling upregulates the expression of spinal inflammatory mediators contributing to nociceptive sensitization in a rodent fracture model of complex regional pain syndrome. PMID:25932690

  9. Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to postfracture nociceptive sensitization.

    PubMed

    Shi, Xiaoyou; Guo, Tian-Zhi; Wei, Tzuping; Li, Wen-Wu; Clark, David J; Kingery, Wade S

    2015-10-01

    Tibia fracture induces exaggerated substance P (SP) and calcitonin gene-related peptide (CGRP) signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hind limbs of rats similar to those seen in complex regional pain syndrome. Inflammatory changes in the spinal cord contribute to nociceptive sensitization in a variety of animal pain models. This study tested the hypothesis that fracture-induced exaggerated neuropeptide signaling upregulates spinal inflammatory mediator expression, leading to postfracture hind limb nociceptive sensitization. At 4 weeks after performing tibia fracture and casting in rats, we measured hind limb allodynia, unweighting, warmth, edema, and spinal cord neuropeptide and inflammatory mediator content. The antinociceptive effects of intrathecally injected neuropeptide and inflammatory mediator receptor antagonists were evaluated in fracture rats. Transgenic fracture mice lacking SP or the CGRP RAMP1 receptor were used to determine the effects of neuropeptide signaling on postfracture pain behavior and spinal inflammatory mediator expression. Hind limb allodynia, unweighting, warmth, edema, increased spinal SP and CGRP, and increased spinal inflammatory mediator expression (TNF, IL-1, IL-6, CCL2, and nerve growth factor) were observed at 4 weeks after fracture in rats. Fracture-induced increases in spinal inflammatory mediators were not observed in fracture mice lacking SP or the CGRP receptor, and these mice had attenuated postfracture nociceptive sensitization. Intrathecal injection of selective receptor antagonists for SP, CGRP, TNF, IL-1, IL-6, CCL2, or nerve growth factor each reduced pain behaviors in the fracture rats. Collectively, these data support the hypothesis that facilitated spinal neuropeptide signaling upregulates the expression of spinal inflammatory mediators contributing to nociceptive sensitization in a rodent fracture model of complex regional pain syndrome.

  10. Phylogeography of E1b1b1b-M81 haplogroup and analysis of its subclades in Morocco.

    PubMed

    Reguig, Ahmed; Harich, Nourdin; Barakat, Abdelhamid; Rouba, Hassan

    2014-01-01

    In this study we analyzed 295 unrelated Berber-speaking men from northern, central, and southern Morocco to characterize frequency of the E1b1b1b-M81 haplogroup and to refine the phylogeny of its subclades: E1b1b1b1-M107, E1b1b1b2-M183, and E1b1b1b2a-M165. For this purpose, we typed four biallelic polymorphisms: M81, M107, M183, and M165. A large majority of the Berber-speaking male lineages belonged to the Y-chromosomal E1b1b1b-M81 haplogroup. The frequency ranged from 79.1% to 98.5% in all localities sampled. E1b1b1b2-M183 was the most dominant subclade in our samples, ranging from 65.1% to 83.1%. In contrast, the E1b1b1b1-M107 and E1b1b1b2a-M165 subclades were not found in our samples. Our results suggest a predominance of the E1b1b1b-M81 haplogroup among Moroccan Berber-speaking males with a decreasing gradient from south to north. The most prevalent subclade in this haplogroup was E1b1b1b2-M183, for which diffferences among these three groups were statistically significant between central and southern groups. PMID:25397701

  11. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis

    PubMed Central

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PMID:27200349

  12. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

    PubMed Central

    Jiang, Huaqi; Grenley, Marc O.; Bravo, Maria-Jose; Blumhagen, Rachel Z.; Edgar, Bruce A.

    2010-01-01

    Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thus maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration following enteric infection by the bacterium, Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis. PMID:21167805

  13. Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs

    PubMed Central

    Scholz, Paul; Mohrhardt, Julia; Jansen, Fabian; Kalbe, Benjamin; Haering, Claudia; Klasen, Katharina; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl− efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca2+ from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner. PMID:27065801

  14. Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

    PubMed

    Scholz, Paul; Mohrhardt, Julia; Jansen, Fabian; Kalbe, Benjamin; Haering, Claudia; Klasen, Katharina; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

  15. Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

    PubMed

    Scholz, Paul; Mohrhardt, Julia; Jansen, Fabian; Kalbe, Benjamin; Haering, Claudia; Klasen, Katharina; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner. PMID:27065801

  16. Lys63-linked polyubiquitination of BRAF at lysine 578 is required for BRAF-mediated signaling

    PubMed Central

    An, Lei; Jia, Wei; Yu, Yang; Zou, Ning; Liang, Li; Zhao, Yanling; Fan, Yihui; Cheng, Jin; Shi, Zhongcheng; Xu, Gufeng; Li, Grace; Yang, Jianhua; Zhang, Hong

    2013-01-01

    The RAF kinase family is essential in mediating signal transduction from RAS to ERK. BRAF constitutively active mutations correlate with human cancer development. However, the precise molecular regulation of BRAF activation is not fully understood. Here we report that BRAF is modified by Lys63-linked polyubiquitination at lysine 578 within its kinase domain once it is activated by gain of constitutively active mutation or epidermal growth factor (EGF) stimulation. Substitution of BRAF lysine 578 with arginine (K578R) inhibited BRAF-mediated ERK activation. Furthermore, ectopic expression of BRAF K578R mutant inhibited anchorage-independent colony formation of MCF7 breast cancer cell line. Our studies have identified a previously unrecognized regulatory role of Lys63-linked polyubiquitination in BRAF-mediated normal and oncogenic signalings. PMID:23907581

  17. Peroxide-dependent MGL sulfenylation regulates 2-AG-mediated endocannabinoid signaling in brain neurons

    PubMed Central

    Dotsey, Emmanuel Y.; Jung, Kwang-Mook; Basit, Abdul; Wei, Don; Daglian, Jennifer; Vacondio, Federica; Armirotti, Andrea; Mor, Marco; Piomelli, Daniele

    2015-01-01

    SUMMARY The second messenger hydrogen peroxide transduces changes in cellular redox state by reversibly oxidizing protein cysteine residues to sulfenic acid. This signaling event regulates many cellular processes, but has been never shown to occur in the brain. Here we report that hydrogen peroxide heightens endocannabinoid signaling in brain neurons through sulfenylation of cysteines C201 and C208 in monoacylglycerol lipase (MGL), a serine hydrolase that deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in nerve terminals. The results suggest that MGL sulfenylation may provide a presynaptic control point for 2-AG-mediated endocannabinoid signaling. PMID:26000748

  18. Peroxide-Dependent MGL Sulfenylation Regulates 2-AG-Mediated Endocannabinoid Signaling in Brain Neurons.

    PubMed

    Dotsey, Emmanuel Y; Jung, Kwang-Mook; Basit, Abdul; Wei, Don; Daglian, Jennifer; Vacondio, Federica; Armirotti, Andrea; Mor, Marco; Piomelli, Daniele

    2015-05-21

    The second messenger hydrogen peroxide transduces changes in the cellular redox state by reversibly oxidizing protein cysteine residues to sulfenic acid. This signaling event regulates many cellular processes but has never been shown to occur in the brain. Here, we report that hydrogen peroxide heightens endocannabinoid signaling in brain neurons through sulfenylation of cysteines C201 and C208 in monoacylglycerol lipase (MGL), a serine hydrolase that deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in nerve terminals. The results suggest that MGL sulfenylation may provide a presynaptic control point for 2-AG-mediated endocannabinoid signaling.

  19. Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma.

    PubMed

    Mu, Xiaodong; Agarwal, Rashmi; March, Daniel; Rothenberg, Adam; Voigt, Clifford; Tebbets, Jessica; Huard, Johnny; Weiss, Kurt

    2016-01-01

    Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia. PMID:27378829

  20. Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma

    PubMed Central

    Agarwal, Rashmi; March, Daniel; Voigt, Clifford

    2016-01-01

    Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia. PMID:27378829

  1. Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells

    PubMed Central

    Ha, Sun-Hyung; Lee, Ji-Min; Kwon, Kyung-Min; Kwak, Choong-Hwan; Abekura, Fukushi; Park, Jun-Young; Cho, Seung-Hak; Lee, Kichoon; Chang, Young-Chae; Lee, Young-Choon; Choi, Hee-Jung; Chung, Tae-Wook; Ha, Ki-Tae; Chang, Hyeun-Wook; Kim, Cheorl-Ho

    2016-01-01

    Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: β1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis. PMID:27144558

  2. Dopaminergic signaling mediates the motivational response underlying the opponent process to chronic but not acute nicotine.

    PubMed

    Grieder, Taryn E; Sellings, Laurie H; Vargas-Perez, Hector; Ting-A-Kee, Ryan; Siu, Eric C; Tyndale, Rachel F; van der Kooy, Derek

    2010-03-01

    The mesolimbic dopamine (DA) system is implicated in the processing of the positive reinforcing effect of all drugs of abuse, including nicotine. It has been suggested that the dopaminergic system is also involved in the aversive motivational response to drug withdrawal, particularly for opiates, however, the role for dopaminergic signaling in the processing of the negative motivational properties of nicotine withdrawal is largely unknown. We hypothesized that signaling at dopaminergic receptors mediates chronic nicotine withdrawal aversions and that dopaminergic signaling would differentially mediate acute vs dependent nicotine motivation. We report that nicotine-dependent rats and mice showed conditioned place aversions to an environment paired with abstinence from chronic nicotine that were blocked by the DA receptor antagonist alpha-flupenthixol (alpha-flu) and in DA D(2) receptor knockout mice. Conversely, alpha-flu pretreatment had no effect on preferences for an environment paired with abstinence from acute nicotine. Taken together, these results suggest that dopaminergic signaling is necessary for the opponent motivational response to nicotine in dependent, but not non-dependent, rodents. Further, signaling at the DA D(2) receptor is critical in mediating withdrawal aversions in nicotine-dependent animals. We suggest that the alleviation of nicotine withdrawal primarily may be driving nicotine motivation in dependent animals.

  3. Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

    PubMed

    Liu, Bo; Zheng, Yonggang; Yin, Feng; Yu, Jianzhong; Silverman, Neal; Pan, Duojia

    2016-01-28

    The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings. PMID:26824654

  4. Dopaminergic signaling mediates the motivational response underlying the opponent process to chronic but not acute nicotine.

    PubMed

    Grieder, Taryn E; Sellings, Laurie H; Vargas-Perez, Hector; Ting-A-Kee, Ryan; Siu, Eric C; Tyndale, Rachel F; van der Kooy, Derek

    2010-03-01

    The mesolimbic dopamine (DA) system is implicated in the processing of the positive reinforcing effect of all drugs of abuse, including nicotine. It has been suggested that the dopaminergic system is also involved in the aversive motivational response to drug withdrawal, particularly for opiates, however, the role for dopaminergic signaling in the processing of the negative motivational properties of nicotine withdrawal is largely unknown. We hypothesized that signaling at dopaminergic receptors mediates chronic nicotine withdrawal aversions and that dopaminergic signaling would differentially mediate acute vs dependent nicotine motivation. We report that nicotine-dependent rats and mice showed conditioned place aversions to an environment paired with abstinence from chronic nicotine that were blocked by the DA receptor antagonist alpha-flupenthixol (alpha-flu) and in DA D(2) receptor knockout mice. Conversely, alpha-flu pretreatment had no effect on preferences for an environment paired with abstinence from acute nicotine. Taken together, these results suggest that dopaminergic signaling is necessary for the opponent motivational response to nicotine in dependent, but not non-dependent, rodents. Further, signaling at the DA D(2) receptor is critical in mediating withdrawal aversions in nicotine-dependent animals. We suggest that the alleviation of nicotine withdrawal primarily may be driving nicotine motivation in dependent animals. PMID:20032966

  5. ATP oscillations mediate inductive action of FGF and Shh signalling on prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2013-01-01

    Skeletal patterns are prefigured by prechondrogenic condensation. Morphogens such as fibroblast growth factor (FGF) and sonic hedgehog (Shh) specify the skeletal patterns in limb development. However, how morphogens regulate prechondrogenic condensation has remained unclear. Recently, it was demonstrated that synchronized Adenosine triphosphate (ATP) oscillations play a critical role in prechondrogenic condensation. Thus, the present study has focused on whether ATP oscillations mediate the actions of major developmental morphogens such as FGF and Shh on prechondrogenic condensation. It has been shown that both FGF and Shh signalling promoted cellular condensation but not chondrogenic differentiation and also induced ATP oscillations. In addition, blockage of FGF and Shh signalling prevented both ATP oscillations and prechondrogenic condensation. Furthermore, it was found that inhibition of ATP oscillations suppressed FGF/Shh-induced prechondrogenic condensation. These results indicate that ATP oscillations mediate the actions of FGF and Shh signalling on prechondrogenic condensation. This study proposes that morphogens organize skeletal patterns via ATP oscillations.

  6. P2y receptor-mediated angiogenesis via vascular endothelial growth factor receptor 2 signaling.

    PubMed

    Rumjahn, Sharif M; Baldwin, Karla A; Buxton, Iain L O

    2007-01-01

    Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 microM ATP and 10 microM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 microM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis.

  7. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate.

    PubMed

    Salmeen, Annette; Andersen, Jannik N; Myers, Michael P; Meng, Tzu-Ching; Hinks, John A; Tonks, Nicholas K; Barford, David

    2003-06-12

    The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.

  8. PTP1B: a double agent in metabolism and oncogenesis

    PubMed Central

    Yip, Shu-Chin; Saha, Sayanti; Chernoff, Jonathan

    2010-01-01

    PTP1B, a non-transmembrane protein tyrosine phosphatase that has long been studied as a negative regulator of insulin and leptin signaling, has recently received renewed attention as an unexpected positive factor in tumorigenesis. In this review, we highlight how views of this enzyme have evolved from regarding it as a simple metabolic off-switch to a more complex view of PTP1B as an enzyme that can play both negative and positive roles diverse signaling pathways. These dual characteristics make PTP1B a particularly attractive therapeutic target for diabetes, obesity, and perhaps breast cancer. PMID:20381358

  9. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    PubMed

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  10. Role of 5-hydroxytryptamine 1B (5-HT1B) receptors in the regulation of ethanol intake in rodents

    PubMed Central

    Sari, Youssef

    2012-01-01

    Evidence indicates that the serotonergic system is important in mediating dependence on and craving for alcohol. Among serotonin receptors, 5-hydroxytryptamine 1B (5-HT1B) receptors have been associated with drug abuse including alcohol. In this review, the neurocircuitry involving 5-HT1B receptors in central reward brain regions related to alcohol intake are discussed in detail. Emphasis has been placed on the pharmacological manipulations of 5-HT1B receptor-mediated alcohol intake. Furthermore, 5-HT1B auto- and hetero-receptors regulate alcohol intake through the regulatory mechanism involving release of 5-HT, gamma-aminobutyric acid (GABA), dopamine, and glutamate is evaluated. Thus, interactions between 5-HT1B receptors and these neurotransmitter systems are suggested to modulate alcohol-drinking behavior. This review on the role of 5-HT1B receptors in neurotransmitter release and consequent alcohol intake provides important information about the potential therapeutic role of 5-HT1B receptors for the treatment of alcohol dependence. PMID:23118018

  11. M2 Macrophage Polarization Mediates Anti-inflammatory Effects of Endothelial Nitric Oxide Signaling

    PubMed Central

    Lee, Woo Je; Tateya, Sanshiro; Cheng, Andrew M.; Rizzo-DeLeon, Norma; Wang, Nicholas F.; Handa, Priya; Wilson, Carole L.; Clowes, Alexander W.; Sweet, Ian R.; Bomsztyk, Karol; Schwartz, Michael W.

    2015-01-01

    Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance. PMID:25845662

  12. Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.

    PubMed

    Ladbury, John E; Arold, Stefan T

    2011-01-01

    Intracellular signaling from receptor tyrosine kinases (RTK) on extracellular stimulation is fundamental to all cellular processes. The protein-protein interactions which form the basis of this signaling are mediated through a limited number of polypeptide domains. For signal transduction without corruption, based on a model where signaling pathways are considered as linear bimolecular relays, these interactions have to be highly specific. This is particularly the case when one considers that any cell may have copies of similar binding domains found in numerous proteins. In this work, an overview of the thermodynamics of binding of two of the most common of these domains (SH2 and SH3 domains) is given. This, coupled with insight from high-resolution structural detail, provides a comprehensive survey of how recognition of cognate binding sites for these domains occurs. Based on the data presented, we conclude that specificity offered by these interactions of SH2 and SH3 domains is limited and not sufficient to enforce mutual exclusivity in RTK-mediated signaling. This may explain the current lack of success in pharmaceutical intervention to inhibit the interactions of these domains when they are responsible for aberrant signaling and the resulting disease states such as cancer.

  13. Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

    PubMed Central

    2010-01-01

    Background Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable. Results To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response. Conclusions These results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses. PMID:20504319

  14. Vibrio cholerae porin OmpU induces LPS tolerance by attenuating TLR-mediated signaling.

    PubMed

    Sakharwade, Sanica C; Mukhopadhaya, Arunika

    2015-12-01

    Porins can act as pathogen-associated molecular patterns, can be recognized by the host immune system and modulate immune responses. Vibrio choleraeporin OmpU aids in bacterial survival in the human gut by increasing resistance against bile acids and anti-microbial peptides. V. choleraeOmpU is pro-inflammatory in nature. However, interestingly, it can also down-regulate LPS-mediated pro-inflammatory responses. In this study, we have explored how OmpU-pretreatment affects LPS-mediated responses. Our study indicates that OmpU-pretreatment followed by LPS-activation does not induce M2-polarization of macrophages/monocytes. Further, OmpU attenuates LPS-mediated TLR2/TLR6 signaling by decreasing the association of TLRs along with recruitment of MyD88 and IRAKs to the receptor complex. This results in decreased translocation of NFκB in the nucleus. Additionally, OmpU-pretreatment up-regulates expression of IRAK-M, a negative regulator of TLR signaling, in RAW 264.7 mouse macrophage cells upon LPS-stimulation. Suppressor cytokine IL-10 is partially involved in OmpU-induced down-regulation of LPS-mediated TNFα production in human PBMCs. Furthermore, OmpU-pretreatment also affects macrophage function, by enhancing phagocytosis in LPS-treated RAW 264.7 cells, and down-regulates LPS-induced cell surface expression of co-stimulatory molecules. Altogether, OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated TLR signaling pathway.

  15. PKCα-Mediated Signals Regulate the Motile Responses of Cochlear Outer Hair Cells.

    PubMed

    Park, Channy; Kalinec, Federico

    2015-05-01

    There is strong evidence that changes in the actin/spectrin-based cortical cytoskeleton of outer hair cells (OHCs) regulate their motile responses as well as cochlear amplification, the process that optimizes the sensitivity and frequency selectivity of the mammalian inner ear. Since a RhoA/protein kinase C (PKC)-mediated pathway is known to inhibit the actin-spectrin interaction in other cell models, we decided to investigate whether this signaling cascade could also participate in the regulation of OHC motility. We used high-speed video microscopy and confocal microscopy to explore the effects of pharmacological activation of PKCα, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ with lysophosphatidic acid (LPA) and their inhibition with bisindolylmaleimide I, as well as inhibition of RhoA and Rho-associated protein kinase (ROCK) with C3 and Y-27632, respectively. Motile responses were induced in isolated guinea pig OHCs by stimulation with an 8 V/cm external alternating electrical field as 50 Hz bursts of square wave pulses (100 ms on/off). We found that LPA increased expression of PKCα and PKCζ only, with PKCα, but not PKCζ, phosphorylating the cytoskeletal protein adducin of both Ser-726 and Thr-445. Interestingly, however, inhibition of PKCα reduced adducin phosphorylation only at Ser-726. We also determined that LPA activation of a PKCα-mediated signaling pathway simultaneously enhanced OHC electromotile amplitude and cell shortening, and facilitated RhoA/ROCK/LIMK1-mediated cofilin phosphorylation. Altogether, our results suggest that PKCα-mediated signals, probably via adducin-mediated inhibition of actin-spectrin binding and cofilin-mediated depolymerization of actin filaments, play an essential role in the homeostatic regulation of OHC motility and cochlear amplification. PMID:25954875

  16. A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

    PubMed

    Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

    2003-02-28

    Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals. PMID:12446706

  17. A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

    PubMed

    Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

    2003-02-28

    Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

  18. Suppressor of Cytokine Signalling-6 Promotes Neurite Outgrowth via JAK2/STAT5-Mediated Signalling Pathway, Involving Negative Feedback Inhibition

    PubMed Central

    Gupta, Sakshi; Mishra, Kanchan; Surolia, Avadhesha; Banerjee, Kakoli

    2011-01-01

    Background Suppressors of cytokine signalling (SOCS) protein family are key regulators of cellular responses to cytokines and play an important role in the nervous system. The SOCS6 protein, a less extensively studied SOCS family member, has been shown to induce insulin resistance in the retina and promote survival of the retinal neurons. But no reports are available about the role of SOCS6 in neuritogenesis. In this study, we examined the role of SOCS6 in neurite outgrowth and neuronal cell signalling. Methodology/Principal Findings The effect of SOCS6 in neural stem cells differentiation was studied in neural stem cells and PC12 cell line. Highly elevated levels of SOCS6 were found upon neural cell differentiation both at the mRNA and protein level. Furthermore, SOCS6 over-expression lead to increase in neurite outgrowth and degree of branching, whereas SOCS6 knockdown with specific siRNAs, lead to a significant decrease in neurite initiation and extension. Insulin-like growth factor-1 (IGF-1) stimulation which enhanced neurite outgrowth of neural cells resulted in further enhancement of SOCS6 expression. Jak/Stat (Janus Kinase/Signal Transducer And Activator Of Transcription) pathway was found to be involved in the SOCS6 mediated neurite outgrowth. Bioinformatics study revealed presence of putative Stat binding sites in the SOCS6 promoter region. Transcription factors Stat5a and Stat5b were involved in SOCS6 gene upregulation leading to neuronal differentiation. Following differentiation, SOCS6 was found to form a ternary complex with IGFR (Insulin Like Growth Factor-1 Receptor) and JAK2 which acted in a negative feedback loop to inhibit pStat5 activation. Conclusion/Significance The current paradigm for the first time states that SOCS6, a SOCS family member, plays an important role in the process of neuronal differentiation. These findings define a novel molecular mechanism for Jak2/Stat5 mediated SOCS6 signalling. PMID:22125600

  19. The anti-proliferative effect of TI1B, a major Bowman-Birk isoinhibitor from pea (Pisum sativum L.), on HT29 colon cancer cells is mediated through protease inhibition.

    PubMed

    Clemente, Alfonso; Carmen Marín-Manzano, M; Jiménez, Elisabeth; Carmen Arques, M; Domoney, Claire

    2012-08-01

    Bowman-Birk inhibitors (BBI) from legumes, such as soyabean, pea, lentil and chickpea, are naturally occurring plant protease inhibitors which have potential health-promoting properties within the mammalian gastrointestinal tract. BBI can survive both acidic conditions and the action of proteolytic enzymes within the stomach and small intestine, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. In a previous study, we reported the ability of a recombinant form of TI1B (rTI1B), representing a major BBI isoinhibitor from pea, to influence negatively the growth of human colorectal adenocarcinoma HT29 cells in vitro. In the present study, we investigate if this effect is related directly to the intrinsic ability of BBI to inhibit serine proteases. rTI1B and a novel engineered mutant, having amino acid substitutions at the P1 positions in the two inhibitory domains, were expressed in the yeast Pichia pastoris. The rTI1B proved to be active against trypsin and chymotrypsin, showing K i values at nanomolar concentrations, whereas the related mutant protein was inactive against both serine proteases. The proliferation of HT29 colon cancer cells was significantly affected by rTI1B in a dose-dependent manner (IC50 = 31 (sd 7) μm), whereas the inactive mutant did not show any significant effect on colon cancer cell growth. In addition, neither recombinant protein affected the growth of non-malignant colonic fibroblast CCD-18Co cells. These findings suggest that serine proteases should be considered as important targets in investigating the potential chemopreventive role of BBI during the early stages of colorectal carcinogenesis. PMID:22916809

  20. Calmodulin physically interacts with the erythropoietin receptor and enhances Jak2-mediated signaling

    SciTech Connect

    Kakihana, Kazuhiko; Yamamoto, Masahide; Iiyama, Mitsuko; Miura, Osamu . E-mail: miura.hema@tmd.ac.jp

    2005-09-23

    Stimulation of the erythropoietin receptor (EpoR) induces a transient increase in intracellular Ca{sup 2+} level as well as activation of the Jak2 tyrosine kinase to stimulate various downstream signaling pathways. Here, we demonstrate that the universal Ca{sup 2+} receptor calmodulin (CaM) binds EpoR in a Ca{sup 2+}-dependent manner in vitro. Binding studies using various EpoR mutants in hematopoietic cells showed that CaM binds the membrane-proximal 65-amino-acid cytoplasmic region (amino acids 258-312) of EpoR that is critical for activation of Jak2-mediated EpoR signaling. Structurally unrelated CaM antagonists, W-13 and CMZ, inhibited activation of Jak2-mediated EpoR signaling pathways, whereas W-12, a W-13 analog, did not show any significant inhibitory effect. Moreover, overexpression of CaM augmented Epo-induced tyrosine phosphorylation of the EpoR. W-13, but not W-12, also inhibited Epo-induced proliferation and survival. Together, these results indicate that CaM binds to the membrane-proximal EpoR cytoplasmic region and plays an essential role in activation of Jak2-mediated EpoR signaling.

  1. 15-oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways

    PubMed Central

    Snyder, Nathaniel W.; Golin-Bisello, Franca; Gao, Yang; Blair, Ian A.; Freeman, Bruce A.; Wendell, Stacy Gelhaus

    2014-01-01

    Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic “inactivation” of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,β-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKβ inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators. PMID:25450232

  2. Calcineurin/NFAT signaling and innate host defence: a role for NOD1-mediated phagocytic functions

    PubMed Central

    2014-01-01

    The calcineurin/nuclear factor of activated T cells (NFATs) signaling pathway plays a central role in T cell mediated adaptive immune responses, but a number of recent studies demonstrated that calcineurin/NFAT signaling also plays a key role in the control of the innate immune response by myeloid cells. Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and in a variety of immune diseases. These immunosuppressive drugs have adverse effects and significantly increase host’s susceptibility towards bacterial or fungal infections. Recent studies highlighted the role of NFAT signaling in fungal infection and in the control of the pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which predominantly senses invasive Gram-negative bacteria and mediates neutrophil phagocytic functions. This review summarises some of the current knowledge concerning the role of NFAT signaling in the innate immune response and the recent advances on NFAT-dependent inhibition of NOD1-mediated innate immune response caused by CsA, which may contribute to sensitizing transplant recipients to bacterial infection. PMID:24479879

  3. Role of CD137 signaling in dengue virus-mediated apoptosis

    SciTech Connect

    Nagila, Amar; Netsawang, Janjuree; Srisawat, Chatchawan; Noisakran, Sansanee; Morchang, Atthapan; Yasamut, Umpa; Puttikhunt, Chunya; Kasinrerk, Watchara; and others

    2011-07-08

    Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  4. Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants

    PubMed Central

    Samanta, Subhasis; Thakur, Jitendra K.

    2015-01-01

    Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes. PMID:26442070

  5. Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants.

    PubMed

    Samanta, Subhasis; Thakur, Jitendra K

    2015-01-01

    Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.

  6. ROP GTPase-mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana.

    PubMed

    Lin, Deshu; Ren, Huibo; Fu, Ying

    2015-01-01

    In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell-to-cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP-mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin-ROP signaling networks have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes. Here, we review findings related to the discovery of this novel auxin-signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well-known Transport Inhibitor Response (TIR)-Auxin Signaling F-Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane-localized, transmembrane kinase (TMK) receptor-like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self-organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis.

  7. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

    PubMed

    Kringelbach, Tina M; Aslan, Derya; Novak, Ivana; Ellegaard, Maria; Syberg, Susanne; Andersen, Christina K B; Kristiansen, Kim A; Vang, Ole; Schwarz, Peter; Jørgensen, Niklas R

    2015-12-01

    Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction.

  8. Feedback Regulation of Cell-Substratum Adhesion by Integrin-Mediated Intracellular Ca2+ Signaling

    NASA Astrophysics Data System (ADS)

    Sjaastad, Michael D.; Angres, Brigitte; Lewis, Richard S.; Nelson, W. James

    1994-08-01

    Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, wound healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion

  9. Central amygdala PKC-δ+ neurons mediate the influence of multiple anorexigenic signals

    PubMed Central

    Cai, Haijiang; Haubensak, Wulf; Anthony, Todd; Anderson, David J

    2014-01-01

    Feeding can be inhibited by multiple cues, including those associated with satiety, sickness or unpalatable food. How such anorexigenic signals inhibit feeding at the neural circuit level is incompletely understood. While some inhibitory circuits have been identified, it is not yet clear whether distinct anorexigenic influences are processed in a convergent or parallel manner. The amygdala central nucleus (CEA) has been implicated in feeding control, but its role is controversial. The lateral subdivision of CEA (CEl) contains a subpopulation of GABAergic neurons, marked by protein kinase C-δ. Here we show that CEl PKC-δ+ neurons in mice are activated by diverse anorexigenic signals in vivo, required for the inhibition of feeding by such signals, and strongly suppress food intake when activated. They receive pre-synaptic inputs from anatomically distributed neurons activated by different anorexigenic agents. These data suggest that CEl PKC-δ+ neurons constitute an important node that mediates the influence of multiple anorexigenic signals. PMID:25064852

  10. Hippo signaling mediators Yap and Taz are required in the epicardium for coronary vasculature development

    PubMed Central

    Singh, Anamika; Ramesh, Sindhu; Cibi, Dasan Mary; Yun, Lim Sze; Li, Jun; Li, Li; Manderfield, Lauren J.; Olson, Eric N.; Epstein, Jonathan A.; Singh, Manvendra K.

    2016-01-01

    Summary Formation of the coronary vasculature is a complex and precisely coordinated morphogenetic process that begins with the formation of epicardium. The epicardium gives rise to many components of the coronary vasculature, including fibroblasts, smooth muscle cells and endothelium. Hippo signaling components have been implicated in cardiac development and regeneration. However a role of Hippo signaling in the epicardium has not been explored. Employing a combination of genetic and pharmacological approaches, we demonstrate that inhibition of Hippo signaling mediators Yap and Taz leads to impaired epicardial epithelial-to-mesenchymal transition (EMT) and a reduction in epicardial cell proliferation and differentiation into coronary endothelial cells. We provide evidence that Yap and Taz control epicardial cell behavior, in part by regulating Tbx18 and Wt1 expression. Our findings show a role for Hippo signaling in epicardial cell proliferation, EMT and cell fate specification during cardiac organogenesis. PMID:27160901

  11. Female Iberian wall lizards prefer male scents that signal a better cell-mediated immune response

    PubMed Central

    López, Pilar; Martín, José

    2005-01-01

    In spite of the importance of chemoreception in sexual selection of lizards, only a few studies have examined the composition of chemical signals, and it is unknown whether and how chemicals provide honest information. Chemical signals might be honest if there were a trade-off between sexual advertisement and the immune system. Here, we show that proportions of cholesta-5,7-dien-3-ol in femoral secretions of male Iberian wall lizards (Podarcis hispanica) were related to their T-cell-mediated immune response. Thus, only males with a good immune system may allocate higher amounts of this chemical to signalling. Furthermore, females selected scents of males with higher proportions of cholesta-5,7-dien-3-ol and lower proportions of cholesterol. Thus, females might base their mate choice on the males' quality as indicated by the composition of their chemical signals. PMID:17148218

  12. SPATA2-Mediated Binding of CYLD to HOIP Enables CYLD Recruitment to Signaling Complexes.

    PubMed

    Kupka, Sebastian; De Miguel, Diego; Draber, Peter; Martino, Luigi; Surinova, Silvia; Rittinger, Katrin; Walczak, Henning

    2016-08-30

    Recruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC). Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF), resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes. PMID:27545878

  13. Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.).

    PubMed

    Xing, Li-Bo; Zhang, Dong; Li, You-Mei; Shen, Ya-Wen; Zhao, Cai-Ping; Ma, Juan-Juan; An, Na; Han, Ming-Yu

    2015-10-01

    Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees. PMID:26412779

  14. Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.).

    PubMed

    Xing, Li-Bo; Zhang, Dong; Li, You-Mei; Shen, Ya-Wen; Zhao, Cai-Ping; Ma, Juan-Juan; An, Na; Han, Ming-Yu

    2015-10-01

    Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees.

  15. Methoxychlor enhances degranulation of murine mast cells by regulating FcεRI-mediated signal transduction.

    PubMed

    Yasunaga, Sho; Nishi, Kosuke; Nishimoto, Sogo; Sugahara, Takuya

    2015-01-01

    Methoxychlor, an organochlorine insecticide developed to replace DDT (dichlorodiphenyltrichloroethane), has been reported to induce mast cell degranulation and to enhance IgE-mediated allergic responses. However, the mechanisms underlying these effects are not clear. To clarify potential mechanisms, the effects of methoxychlor on degranulation of mast cells were examined. Degranulation responses were evaluated using RBL-2H3 cells and mouse bone marrow-derived mast cells with either the antigen-induced or calcium ionophore-induced stimulation. Phosphorylation of enzymes related to signaling events associated with mast cell degranulation was analyzed by immunoblotting. Effects on vascular permeability in the passive cutaneous anaphylaxis reaction were evaluated following oral administration of methoxychlor to BALB/c mice. The results indicated that methoxychlor caused increased mast cell degranulation in the presence of antigen, whereas it had no effect on calcium ionophore-induced degranulation of RBL-2H3 cells. Immunoblot analyses demonstrated that the phosphorylation level of phosphoinositide 3-kinase (which plays a central role in mast cell signaling) was increased by methoxychlor during antigen-induced degranulation. In addition, methoxychlor activated the signaling pathway via the high-affinity IgE receptor by inducing phosphorylation of Syk and PLCγ1/2, which transfer the signal for degranulation downstream. Lastly, oral administration of methoxychlor exhibited a tendency to promote vascular permeability in passive cutaneous anaphylaxis model mice. Taken together, the results here suggested that methoxychlor enhanced degranulation through FcεRI-mediated signaling and promoted allergenic symptoms involved in mast cell degranulation.

  16. Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

    PubMed

    Maity, Gargi; Fahreen, Shabana; Banerji, Aniruddha; Roy Choudhury, Paromita; Sen, Triparna; Dutta, Anindita; Chatterjee, Amitava

    2010-03-01

    Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

  17. Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis

    PubMed Central

    Reis, Carlos R; Chen, Ping-Hung; Srinivasan, Saipraveen; Aguet, François; Mettlen, Marcel; Schmid, Sandra L

    2015-01-01

    Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this “checkpoint.” Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3β signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME. PMID:26139537

  18. '2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

    PubMed

    Roulston, Claire; Luke, Garry A; de Felipe, Pablo; Ruan, Lin; Cope, Jonathan; Nicholson, John; Sukhodub, Andriy; Tilsner, Jens; Ryan, Martin D

    2016-08-01

    We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting. PMID:27161495

  19. Dynamics of ubiquitin-mediated signalling: insights from mathematical modelling and experimental studies.

    PubMed

    Nguyen, Lan K

    2016-05-01

    Post-translational modification of cellular proteins by ubiquitin is a pivotal regulatory event that controls not only protein degradation, but also a variety of non-proteolytic functions. Ubiquitination is involved in a broad array of physiological processes, and its dysregulation has been associated with many human diseases, including neuronal disorders and cancers. Ubiquitin-mediated signalling has thus come to the forefront of biomedical research. It is increasingly apparent that ubiquitination is a highly complex and dynamic process, evidenced by a myriad of ways of ubiquitin chain formation, tightly regulatory mechanisms involving E3 ligases and deubiquitinating enzymes and extensive crosstalk with other post-translational modifications. To unravel the complexity of ubiquitination and understand the dynamic properties of ubiquitin-mediated signalling are challenging, but critical topics in ubiquitin research, which will undoubtedly benefit our effort in developing strategies that could target ubiquitin signalling for therapeutics. Computational modelling and model-based approaches are emerging as promising tools that help tackle the complexity and provide useful frameworks for quantitative and dynamical analysis of ubiquitin signalling. In this article, I will discuss recent advances in our understanding of the dynamic behaviour of ubiquitination from both theoretical and experimental studies, and aspects of ubiquitin signalling that may have major dynamical consequences. It is expected the discussed issues will be of relevant interest to both the ubiquitin and systems biology fields.

  20. Integrin-mediated adhesion complex: Cooption of signaling systems at the dawn of Metazoa.

    PubMed

    Sebé-Pedrós, Arnau; Ruiz-Trillo, Iñaki

    2010-09-01

    The integrin-mediated adhesion machinery is the primary cell-matrix adhesion mechanism in Metazoa. The integrin adhesion complex, which modulates important aspects of the cell physiology, is composed of integrins (alpha and beta subunits) and several scaffolding and signaling proteins. Integrins appeared to be absent in all non-metazoan eukaryotes so-far analyzed, including fungi, plants and choanoflagellates, the sister-group to Metazoa. Thus, integrins and, therefore, the integrin-mediated adhesion and signaling mechanism was considered a metazoan innovation. Recently, a broad comparative genomic analysis including new genome data from several unicellular organisms closely related to fungi and metazoans shattered previous views. The integrin adhesion and signaling complex is not specific to Metazoa, but rather it is present in apusozoans and holozoan protists. Thus, this important signaling and adhesion system predated the origin of Fungi and Metazoa, and was subsequently lost in fungi and choanoflagellates. This finding suggests that cooption played a more important role in the origin of Metazoa than previously believed. Here, we hypothesize that the integrin adhesome was ancestrally involved in signaling.

  1. Crosstalk between Akt/GSK3β signaling and dynamin-1 regulates clathrin-mediated endocytosis.

    PubMed

    Reis, Carlos R; Chen, Ping-Hung; Srinivasan, Saipraveen; Aguet, François; Mettlen, Marcel; Schmid, Sandra L

    2015-08-13

    Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3β signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME.

  2. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  3. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  4. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  5. Charge-signal multiplication mediated by urea wires inside Y-shaped carbon nanotubes

    SciTech Connect

    Lv, Mei; Liu, Zengrong; He, Bing; Xiu, Peng E-mail: ystu@shu.edu.cn; Tu, Yusong E-mail: ystu@shu.edu.cn

    2014-07-28

    In previous studies, we reported molecular dynamics (MD) simulations showing that single-file water wires confined inside Y-shaped single-walled carbon nanotubes (Y-SWNTs) held strong and robust capability to convert and multiply charge signals [Y. S. Tu, P. Xiu, R. Z. Wan, J. Hu, R. H. Zhou, and H. P. Fang, Proc. Natl. Acad. Sci. U.S.A. 106, 18120 (2009); Y. Tu, H. Lu, Y. Zhang, T. Huynh, and R. Zhou, J. Chem. Phys. 138, 015104 (2013)]. It is fascinating to see whether the signal multiplication can be realized by other kinds of polar molecules with larger dipole moments (which make the experimental realization easier). In this article, we use MD simulations to study the urea-mediated signal conversion and multiplication with Y-SWNTs. We observe that when a Y-SWNT with an external charge of magnitude 1.0 e (the model of a signal at the single-electron level) is solvated in 1 M urea solutions, urea can induce drying of the Y-SWNT and fill its interiors in single-file, forming Y-shaped urea wires. The external charge can effectively control the dipole orientation of the urea wire inside the main channel (i.e., the signal can be readily converted), and this signal can further be multiplied into 2 (or more) output signals by modulating dipole orientations of urea wires in bifurcated branch channels of the Y-SWNT. This remarkable signal transduction capability arises from the strong dipole-induced ordering of urea wires under extreme confinement. We also discuss the advantage of urea as compared with water in the signal multiplication, as well as the robustness and biological implications of our findings. This study provides the possibility for multiplying signals by using urea molecules (or other polar organic molecules) with Y-shaped nanochannels and might also help understand the mechanism behind signal conduction in both physical and biological systems.

  6. Charge-signal multiplication mediated by urea wires inside Y-shaped carbon nanotubes.

    PubMed

    Lv, Mei; He, Bing; Liu, Zengrong; Xiu, Peng; Tu, Yusong

    2014-07-28

    In previous studies, we reported molecular dynamics (MD) simulations showing that single-file water wires confined inside Y-shaped single-walled carbon nanotubes (Y-SWNTs) held strong and robust capability to convert and multiply charge signals [Y. S. Tu, P. Xiu, R. Z. Wan, J. Hu, R. H. Zhou, and H. P. Fang, Proc. Natl. Acad. Sci. U.S.A. 106, 18120 (2009); Y. Tu, H. Lu, Y. Zhang, T. Huynh, and R. Zhou, J. Chem. Phys. 138, 015104 (2013)]. It is fascinating to see whether the signal multiplication can be realized by other kinds of polar molecules with larger dipole moments (which make the experimental realization easier). In this article, we use MD simulations to study the urea-mediated signal conversion and multiplication with Y-SWNTs. We observe that when a Y-SWNT with an external charge of magnitude 1.0 e (the model of a signal at the single-electron level) is solvated in 1 M urea solutions, urea can induce drying of the Y-SWNT and fill its interiors in single-file, forming Y-shaped urea wires. The external charge can effectively control the dipole orientation of the urea wire inside the main channel (i.e., the signal can be readily converted), and this signal can further be multiplied into 2 (or more) output signals by modulating dipole orientations of urea wires in bifurcated branch channels of the Y-SWNT. This remarkable signal transduction capability arises from the strong dipole-induced ordering of urea wires under extreme confinement. We also discuss the advantage of urea as compared with water in the signal multiplication, as well as the robustness and biological implications of our findings. This study provides the possibility for multiplying signals by using urea molecules (or other polar organic molecules) with Y-shaped nanochannels and might also help understand the mechanism behind signal conduction in both physical and biological systems.

  7. Charge-signal multiplication mediated by urea wires inside Y-shaped carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Lv, Mei; He, Bing; Liu, Zengrong; Xiu, Peng; Tu, Yusong

    2014-07-01

    In previous studies, we reported molecular dynamics (MD) simulations showing that single-file water wires confined inside Y-shaped single-walled carbon nanotubes (Y-SWNTs) held strong and robust capability to convert and multiply charge signals [Y. S. Tu, P. Xiu, R. Z. Wan, J. Hu, R. H. Zhou, and H. P. Fang, Proc. Natl. Acad. Sci. U.S.A. 106, 18120 (2009); Y. Tu, H. Lu, Y. Zhang, T. Huynh, and R. Zhou, J. Chem. Phys. 138, 015104 (2013)]. It is fascinating to see whether the signal multiplication can be realized by other kinds of polar molecules with larger dipole moments (which make the experimental realization easier). In this article, we use MD simulations to study the urea-mediated signal conversion and multiplication with Y-SWNTs. We observe that when a Y-SWNT with an external charge of magnitude 1.0 e (the model of a signal at the single-electron level) is solvated in 1 M urea solutions, urea can induce drying of the Y-SWNT and fill its interiors in single-file, forming Y-shaped urea wires. The external charge can effectively control the dipole orientation of the urea wire inside the main channel (i.e., the signal can be readily converted), and this signal can further be multiplied into 2 (or more) output signals by modulating dipole orientations of urea wires in bifurcated branch channels of the Y-SWNT. This remarkable signal transduction capability arises from the strong dipole-induced ordering of urea wires under extreme confinement. We also discuss the advantage of urea as compared with water in the signal multiplication, as well as the robustness and biological implications of our findings. This study provides the possibility for multiplying signals by using urea molecules (or other polar organic molecules) with Y-shaped nanochannels and might also help understand the mechanism behind signal conduction in both physical and biological systems.

  8. Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

    PubMed

    Liu, Jing; Lange, Miles D; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R A; Zemlin, Michael; Burrows, Peter D; Su, Kaihong; Carter, Robert H; Zhang, Zhixin

    2013-06-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

  9. Diet-induced obesity mediated by the JNK/DIO2 signal transduction pathway

    PubMed Central

    Vernia, Santiago; Cavanagh-Kyros, Julie; Barrett, Tamera; Jung, Dae Young; Kim, Jason K.; Davis, Roger J.

    2013-01-01

    The cJun N-terminal kinase (JNK) signaling pathway is a key mediator of metabolic stress responses caused by consuming a high-fat diet, including the development of obesity. To test the role of JNK, we examined diet-induced obesity in mice with targeted ablation of Jnk genes in the anterior pituitary gland. These mice exhibited an increase in the pituitary expression of thyroid-stimulating hormone (TSH), an increase in the blood concentration of thyroid hormone (T4), increased energy expenditure, and markedly reduced obesity compared with control mice. The increased amount of pituitary TSH was caused by reduced expression of type 2 iodothyronine deiodinase (Dio2), a gene that is required for T4-mediated negative feedback regulation of TSH expression. These data establish a molecular mechanism that accounts for the regulation of energy expenditure and the development of obesity by the JNK signaling pathway. PMID:24186979

  10. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    SciTech Connect

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. Black-Right-Pointing-Pointer Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. Black-Right-Pointing-Pointer Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. Black-Right-Pointing-Pointer Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through {beta}-catenin-dependent canonical and {beta}-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent

  11. Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation.

    PubMed

    Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

    2015-07-01

    In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

  12. Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

    NASA Astrophysics Data System (ADS)

    Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

    2015-07-01

    In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

  13. Determination of the Role of CBP- and p300-Mediated Wnt Signaling on Colonic Cells

    PubMed Central

    Lazarova, Darina Lazarova

    2016-01-01

    Background The Wnt signaling pathway, mediated through active beta-catenin, is responsible for initiating the majority of cases of human colorectal cancer (CRC), and we have previously shown that hyperactivation of this pathway by histone deacetylase inhibitors (HDACis), such as butyrate, can induce the death of CRC cells. An important cellular switch that mediates the effects of Wnt-signaling activation is variation in the association between beta-catenin and the transcriptional coactivators cAMP response element binding (CREB) binding protein (CBP) and p300. Association of CBP with beta-catenin is thought to activate a set of genes linked to cell proliferation, while the p300-mediated Wnt genetic program is believed to promote cell differentiation. Small molecule agents have been discovered that modulate CBP/p300 Wnt transcriptional programs by altering the association of CBP and p300 to beta-catenin. ICG-001 and ICG-427 inhibit CBP- and p300-mediated Wnt activity, respectively, while IQ-1 prevents the shift from CBP-mediated to a p300-mediated Wnt activity. Objective Aim 1 of this proposal is designed to determine the role of CBP- and p300-mediated Wnt signaling in the response of CRC cells to HDACis. Aim 2 is to determine the role of CBP and p300 in the maintenance of high- and low-Wnt fractions in CRC cell line. Aim 3 will compare the effects of CBP- and p300-mediated Wnt activity on CRC initiation and progression. Methods In Aim 1, cells will be cotreated with HDACis and ICG-001, ICG-427, or IQ-1 and the levels of Wnt activity, apoptosis, proliferation, differentiation, and CBP- or p300-beta-catenin binding measured. Aim 2 of this proposal may mirror similar heterogeneity observed in human tumors and which may be of clinical significance. Aim 3 will use CRC cell line model systems of initiation and progression: the normal colon cell lines CCD-841CoN, the adenoma line LT97, the primary colon carcinoma cell line SW480, and the lymph node metastasis cell line SW

  14. Augmentation of Antigen Receptor–mediated Responses by Histamine H1 Receptor Signaling

    PubMed Central

    Banu, Yasmin; Watanabe, Takeshi

    1999-01-01

    Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein–coupled receptors. Here, we report that histamine may affect antigen receptor–mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative responses of anti-CD3ε– or anti-IgM–stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit–positive cells were depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T cells had low proliferative responses to anti-CD3ε cross-linking or antigen stimulation in vitro. B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor–mediated immune responses, suggesting cross-talk between G protein–coupled receptors and antigen receptor–mediated signaling. PMID:9989982

  15. Fas- and tumor necrosis factor-mediated apoptosis uses the same binding surface of FADD to trigger signal transduction. A typical model for convergent signal transduction.

    PubMed

    Bang, S; Jeong, E J; Kim, I K; Jung, Y K; Kim, K S

    2000-11-17

    FADD is known to function as a common signaling conduit in Fas- and tumor necrosis factor (TNF)-mediated apoptosis. The convergent death signals from the Fas receptor and TNF receptor 1 are transferred to FADD by death domain interactions triggering the same cellular event, caspase-8 activation. In this work, we investigated whether the same binding surface of FADD is used for both signaling pathways by using FADD death domain mutants. Mutations in helices alpha2 and alpha3 of the FADD death domain, the interacting surface with the Fas death domain, affected TNF-mediated apoptosis to various extents. This indicated that TNF-mediated apoptosis uses the same binding surface of the FADD death domain as Fas-mediated apoptosis. The binding specificity is not the same, however. Some mutations affected the binding affinity of the Fas death domain for the FADD death domain, but did not influence TNF-mediated apoptosis and vice versa. Interestingly, all mutants tested that affected TNF-mediated apoptosis have structural perturbations, implying that the structural integrity, involving helices alpha2 and alpha3 in particular, is critical in TNF-mediated apoptosis. Our results suggest that different signaling molecules use a similar structural interaction to trigger the same cellular event, such as caspase-8 recruitment, which could be typical in convergent signal transduction.

  16. Development of an Imaging Mass Spectrometry Technique for Visualizing Localized Cellular Signaling Mediators in Tissues

    PubMed Central

    Sugiura, Yuki; Honda, Kurara; Suematsu, Makoto

    2015-01-01

    In vivo concentrations of cellular signaling mediators such as inflammatory mediators are normally maintained at very low levels due to their strong ability to induce a biological response. The production, diffusion, and decomposition of such mediators are spatio-temporally regulated. Therefore, in order to understand biochemical basis of disease progression and develop new therapeutic strategies, it is important to understand the spatiotemporal dynamics of the signaling mediators in vivo, during the progression of disorders, e.g., chronic inflammatory diseases; however, the lack of effective imaging technology has made it difficult to determine their localizations in vivo. Such characterization requires technical breakthroughs, including molecular imaging methods that are sensitive enough to detect low levels of metabolites in the heterogeneous tissue regions in diseased organs. We and other groups have attempted to fill this technical gap by developing highly sensitive imaging mass spectrometry (IMS) technologies. To date, we have established two key techniques toward this goal, including (i) a sample preparation procedure that has eliminated the problem of the postmortem degradation of labile metabolites, and (ii) on-tissue derivatization of metabolites, which can enhance analyte ionization efficiency. Here, we review recent progress in the development of these technologies as well as how the highly sensitive IMS technique has contributed to increasing understanding of the biochemical basis of disease mechanisms, discovery of new diagnostic markers, and development of new therapies. PMID:26819911

  17. Characterization of Receptor-Mediated Signal Transduction by Escherichia coli Type IIa Heat-Labile Enterotoxin in the Polarized Human Intestinal Cell Line T84

    PubMed Central

    Wimer-Mackin, Susan; Holmes, Randall K.; Wolf, Anne A.; Lencer, Wayne I.; Jobling, Michael G.

    2001-01-01

    Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl− secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl− secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl− secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl− secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli. PMID:11705889

  18. Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

    NASA Astrophysics Data System (ADS)

    Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

    2016-04-01

    Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis.

  19. Oviductal estrogen receptor α signaling prevents protease-mediated embryo death

    PubMed Central

    Winuthayanon, Wipawee; Bernhardt, Miranda L; Padilla-Banks, Elizabeth; Myers, Page H; Edin, Matthew L; Lih, Fred B; Hewitt, Sylvia C; Korach, Kenneth S; Williams, Carmen J

    2015-01-01

    Development of uterine endometrial receptivity for implantation is orchestrated by cyclic steroid hormone-mediated signals. It is unknown if these signals are necessary for oviduct function in supporting fertilization and preimplantation development. Here we show that conditional knockout (cKO) mice lacking estrogen receptor α (ERα) in oviduct and uterine epithelial cells have impaired fertilization due to a dramatic reduction in sperm migration. In addition, all successfully fertilized eggs die before the 2-cell stage due to persistence of secreted innate immune mediators including proteases. Elevated protease activity in cKO oviducts causes premature degradation of the zona pellucida and embryo lysis, and wild-type embryos transferred into cKO oviducts fail to develop normally unless rescued by concomitant transfer of protease inhibitors. Thus, suppression of oviductal protease activity mediated by estrogen-epithelial ERα signaling is required for fertilization and preimplantation embryo development. These findings have implications for human infertility and post-coital contraception. DOI: http://dx.doi.org/10.7554/eLife.10453.001 PMID:26623518

  20. Potentiation of μ–opioid receptor–mediated signaling by ketamine

    PubMed Central

    Gupta, Achla; Devi, Lakshmi A.; Gomes, Ivone

    2013-01-01

    Ketamine, a clinically relevant drug, has been shown to enhance opioid-induced analgesia and prevent hyperalgesia. However, the molecular mechanisms involved are not clearly understood. As previous studies found that activation of opioid receptors leads to the phosphorylation of mitogen-activated protein kinases, we investigated whether ketamine could modulate μ-opioid receptor (μOR)-mediated ERK1/2 phosphorylation. We find that acute treatment with ketamine enhances (~2- to 3-fold) the levels of opioid-induced ERK1/2 phosphorylation in recombinant as well as cells endogenously expressing μOR. Interestingly, we find that in the absence of ketamine ERK1/2 signaling is desensitized 10 min after opioid exposure whereas in its presence significant levels (~3-fold over basal) are detected. In addition, ketamine increases the rate of resensitization of opioid-mediated ERK1/2 signaling (15 min in its presence vs. 30 min in its absence). These results suggest that ketamine increases the effectiveness of opiate-induced signaling by affecting multiple mechanisms. In addition, these effects are observed in heterologous cells expressing μOR suggesting a non-NMDA receptor-mediated action of ketamine. Together this could, in part, account for the observed effects of ketamine on the enhancement of the analgesic effects of opiates as well as in the duration of opiate-induced analgesia. PMID:21692801

  1. Plant signaling networks involving Ca2+ and Rboh/Nox-mediated ROS production under salinity stress

    PubMed Central

    Kurusu, Takamitsu; Kuchitsu, Kazuyuki; Tada, Yuichi

    2015-01-01

    Salinity stress, which induces both ionic and osmotic damage, impairs plant growth and causes severe reductions in crop yield. Plants are equipped with defense responses against salinity stress such as regulation of ion transport including Na+ and K+, accumulation of compatible solutes and stress-related gene expression. The initial Ca2+ influx mediated by plasma membrane ion channels has been suggested to be crucial for the adaptive signaling. NADPH oxidase (Nox)-mediated production of reactive oxygen species (ROS) has also been suggested to play crucial roles in regulating adaptation to salinity stress in several plant species including halophytes. Respiratory burst oxidase homolog (Rboh) proteins show the ROS-producing Nox activity, which are synergistically activated by the binding of Ca2+ to EF-hand motifs as well as Ca2+-dependent phosphorylation. We herein review molecular identity, structural features and roles of the Ca2+-permeable channels involved in early salinity and osmotic signaling, and comparatively discuss the interrelationships among spatiotemporal dynamic changes in cytosolic concentrations of free Ca2+, Rboh-mediated ROS production, and downstream signaling events during salinity adaptation in planta. PMID:26113854

  2. Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction.

    PubMed

    Groeger, Gillian; Nobes, Catherine D

    2007-05-15

    Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganizations, which in turn are partially mediated by Rho-ROCK (Rho kinase) signalling, driving actomyosin contractility. In the present study, we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 (cell division cycle 42) and its effector MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrinB2 triggers rapid, but transient, cell retraction. We show that, although membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting that there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction, but is essential for subsequent Rac-mediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction-recovery responses.

  3. A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

    PubMed Central

    Gonzalez, Nicola H.; Felsner, Gregor; Schramm, Frederic D.; Klingl, Andreas; Maier, Uwe-G.; Bolte, Kathrin

    2011-01-01

    Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1. PMID:21966495

  4. Beta-catenin signaling mediates CD4 expression on mature CD8+ T cells.

    PubMed

    Schenkel, Jason M; Zloza, Andrew; Li, Wei; Narasipura, Srinivas D; Al-Harthi, Lena

    2010-08-15

    Upon activation, a subset of mature human CD8(+) T cells re-expresses CD4 dimly. This CD4(dim)CD8(bright) T cell population is genuine and enriched in antiviral CD8(+) T cell responses. The signaling pathway that leads to CD4 re-expression on mature CD8(+) T cells is not clear. Given that Wnt/beta-catenin signaling plays a critical role in the transition of CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes, we determined whether beta-catenin mediates CD4 expression on mature CD8(+) T cells. We demonstrate that active beta-catenin expression is 20-fold higher on CD4(dim)CD8(bright) than CD4(-)CD8(+) T cells. Activation of beta-catenin signaling, through LiCl or transfection with a constitutively active construct of beta-catenin, induced CD4 on CD8(+) T cells by approximately 10-fold. Conversely, inhibition of beta-catenin signaling through transfection with a dominant-negative construct for T cell factor-4, a downstream effector of beta-catenin signaling, diminished CD4 expression on CD8(+) T cells by 50% in response to T cell activation. Beta-catenin-mediated induction of CD4 on CD8(+) T cells is transcriptionally regulated, as it induced CD4 mRNA, and T cell factor/lymphoid enhancer factor sites were identified within the human CD4 promoter. Further, beta-catenin expression induced the antiapoptotic factor BcL-xL, suggesting that beta-catenin may mediate protection against activation-induced cell death. Collectively, these data demonstrate that beta-catenin is critical in inducing CD4 expression on mature CD8(+) T cells, suggesting that it is a common pathway for CD4 upregulation among thymocytes and mature CD8(+) T cells. PMID:20631314

  5. Protein tyrosine phosphatase 1B and insulin resistance: role of endoplasmic reticulum stress/reactive oxygen species/nuclear factor kappa B axis.

    PubMed

    Panzhinskiy, Evgeniy; Ren, Jun; Nair, Sreejayan

    2013-01-01

    Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [(3)H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.

  6. Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes

    PubMed Central

    Fatima, Tahira; Topuz, Muhamet; Bernadec, Anne; Sicher, Richard; Handa, Avtar K.; Mattoo, Autar K.

    2016-01-01

    of ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon. PMID:27446131

  7. Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes.

    PubMed

    Goyal, Ravinder K; Fatima, Tahira; Topuz, Muhamet; Bernadec, Anne; Sicher, Richard; Handa, Avtar K; Mattoo, Autar K

    2016-01-01

    ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon. PMID:27446131

  8. Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes.

    PubMed

    Goyal, Ravinder K; Fatima, Tahira; Topuz, Muhamet; Bernadec, Anne; Sicher, Richard; Handa, Avtar K; Mattoo, Autar K

    2016-01-01

    ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon.

  9. Protein tyrosine phosphatase 1B inhibitors: a molecular level legitimate approach for the management of diabetes mellitus.

    PubMed

    Thareja, Suresh; Aggarwal, Saurabh; Bhardwaj, T R; Kumar, Manoj

    2012-05-01

    Diabetes mellitus is a systemic disease responsible for morbidity in the western world and is gradually becoming prevalent in developing countries too. The prevalence of diabetes is rapidly increasing in industrialized countries and type 2 diabetes accounts for 90% of the disease. Insulin resistance is a major pathophysiological factor in the development of type 2 diabetes, occurring mainly in muscle, adipose tissues, and liver leading to reduced glucose uptake and utilization and increased glucose production. The prevalence and rising incidence of diabetes emphasized the need to explore new molecular targets and strategies to develop novel antihyperglycemic agents. Protein Tyrosine Phosphatase 1B (PTP 1B) has recently emerged as a promising molecular level legitimate therapeutic target in the effective management of type 2 diabetes. PTP 1B, a cytosolic nonreceptor PTPase, has been implicated as a negative regulator of insulin signal transduction. Therefore, PTP 1B inhibitors would increase insulin sensitivity by blocking the PTP 1B-mediated negative insulin signaling pathway and might be an attractive target for type 2 diabetes mellitus and obesity. With X-ray crystallography and NMR-based fragment screening, the binding interactions of several classes of inhibitors have been elucidated, which could help the design of future PTP 1B inhibitors. The drug discovery research in PTP 1B is a challenging area to work with and many pharmaceutical organizations and academic research laboratories are focusing their research toward the development of potential PTP 1B inhibitors which would prove to be a milestone for the management of diabetes.

  10. Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death.

    PubMed

    Sánchez, Dulfary; Rojas, Mauricio; Hernández, Israel; Radzioch, Danuta; García, Luis F; Barrera, Luis F

    2010-01-01

    Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-kappaB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.

  11. Syndecan-4 negatively regulates antiviral signalling by mediating RIG-I deubiquitination via CYLD

    PubMed Central

    Lin, Wei; Zhang, Jing; Lin, Haiyan; Li, Zexing; Sun, Xiaofeng; Xin, Di; Yang, Meng; Sun, Liwei; Li, Lin; Wang, Hongmei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Retinoic acid-inducible gene I (RIG-I) plays important roles in pathogen recognition and antiviral signalling transduction. Here we show that syndecan-4 (SDC4) is a RIG-I-interacting partner identified in a yeast two-hybrid screen. We find that SDC4 negatively regulates the RIG-I-mediated antiviral signalling in a feedback-loop control manner. The genetic evidence obtained by using knockout mice further emphasizes this biological role of SDC4 in antiviral signalling. Mechanistically, we show that SDC4 interacts with both RIG-I and deubiquitinase CYLD via its carboxyl-terminal intracellular region. SDC4 likely promotes redistribution of RIG-I and CYLD in a perinuclear pattern post viral infection, and thus enhances the RIG-I–CYLD interaction and potentiates the K63-linked deubiquitination of RIG-I. Collectively, our findings uncover a mechanism by which SDC4 antagonizes the activation of RIG-I in a CYLD-mediated deubiquitination-dependent process, thereby balancing antiviral signalling to avoid deleterious effects on host cells. PMID:27279133

  12. Signal integration by lipid-mediated spatial cross talk between Ras nanoclusters.

    PubMed

    Zhou, Yong; Liang, Hong; Rodkey, Travis; Ariotti, Nicholas; Parton, Robert G; Hancock, John F

    2014-03-01

    Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing.

  13. Syndecan-4 negatively regulates antiviral signalling by mediating RIG-I deubiquitination via CYLD.

    PubMed

    Lin, Wei; Zhang, Jing; Lin, Haiyan; Li, Zexing; Sun, Xiaofeng; Xin, Di; Yang, Meng; Sun, Liwei; Li, Lin; Wang, Hongmei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Retinoic acid-inducible gene I (RIG-I) plays important roles in pathogen recognition and antiviral signalling transduction. Here we show that syndecan-4 (SDC4) is a RIG-I-interacting partner identified in a yeast two-hybrid screen. We find that SDC4 negatively regulates the RIG-I-mediated antiviral signalling in a feedback-loop control manner. The genetic evidence obtained by using knockout mice further emphasizes this biological role of SDC4 in antiviral signalling. Mechanistically, we show that SDC4 interacts with both RIG-I and deubiquitinase CYLD via its carboxyl-terminal intracellular region. SDC4 likely promotes redistribution of RIG-I and CYLD in a perinuclear pattern post viral infection, and thus enhances the RIG-I-CYLD interaction and potentiates the K63-linked deubiquitination of RIG-I. Collectively, our findings uncover a mechanism by which SDC4 antagonizes the activation of RIG-I in a CYLD-mediated deubiquitination-dependent process, thereby balancing antiviral signalling to avoid deleterious effects on host cells. PMID:27279133

  14. Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling

    PubMed Central

    Hirata, Shinji; Takayama, Naoya; Jono-Ohnishi, Ryoko; Endo, Hiroshi; Nakamura, Sou; Dohda, Takeaki; Nishi, Masanori; Hamazaki, Yuhei; Ishii, Ei-ichi; Kaneko, Shin; Otsu, Makoto; Nakauchi, Hiromitsu; Kunishima, Shinji; Eto, Koji

    2013-01-01

    Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptor–mediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl–/– mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34+ multipotent hematopoietic progenitor (MPP) population and development of the CD41+GPA+ megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34+ hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC–derived CD34+ HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT. PMID:23908116

  15. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  16. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  17. Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins1

    PubMed Central

    Hewezi, Tarek

    2015-01-01

    Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. PMID:26315856

  18. Antidepressant effects of sleep deprivation require astrocyte-dependent adenosine mediated signaling.

    PubMed

    Hines, D J; Schmitt, L I; Hines, R M; Moss, S J; Haydon, P G

    2013-01-01

    Major depressive disorder is a debilitating condition with a lifetime risk of ten percent. Most treatments take several weeks to achieve clinical efficacy, limiting the ability to bring instant relief needed in psychiatric emergencies. One intervention that rapidly alleviates depressive symptoms is sleep deprivation; however, its mechanism of action is unknown. Astrocytes regulate responses to sleep deprivation, raising the possibility that glial signaling mediates antidepressive-like actions of sleep deprivation. Here, we found that astrocytic signaling to adenosine (A1) receptors was required for the robust reduction of depressive-like behaviors following 12 hours of sleep deprivation. As sleep deprivation activates synaptic A1 receptors, we mimicked the effect of sleep deprivation on depression phenotypes by administration of the A1 agonist CCPA. These results provide the first mechanistic insight into how sleep deprivation impacts mood, and provide a novel pathway for rapid antidepressant development by modulation of glial signaling in the brain. PMID:23321809

  19. The complexity of signalling mediated by the glucagon-like peptide-1 receptor.

    PubMed

    Fletcher, Madeleine M; Halls, Michelle L; Christopoulos, Arthur; Sexton, Patrick M; Wootten, Denise

    2016-04-15

    The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR that is a major therapeutic target for the treatment of type 2 diabetes. The receptor is activated by the incretin peptide GLP-1 promoting a broad range of physiological effects including glucose-dependent insulin secretion and biosynthesis, improved insulin sensitivity of peripheral tissues, preservation of β-cell mass and weight loss, all of which are beneficial in the treatment of type 2 diabetes. Despite this, existing knowledge surrounding the underlying signalling mechanisms responsible for the physiological actions downstream of GLP-1R activation is limited. Here, we review the current understanding around GLP-1R-mediated signalling, in particular highlighting recent contributions to the field on biased agonism, the spatial and temporal aspects for the control of signalling and how these concepts may influence future drug development. PMID:27068973

  20. Asymmetric homotypic interactions of the atypical cadherin flamingo mediate intercellular polarity signaling.

    PubMed

    Chen, Wei-Shen; Antic, Dragana; Matis, Maja; Logan, Catriona Y; Povelones, Michael; Anderson, Graham A; Nusse, Roel; Axelrod, Jeffrey D

    2008-06-13

    Acquisition of planar cell polarity (PCP) in epithelia involves intercellular communication, during which cells align their polarity with that of their neighbors. The transmembrane proteins Frizzled (Fz) and Van Gogh (Vang) are essential components of the intercellular communication mechanism, as loss of either strongly perturbs the polarity of neighboring cells. How Fz and Vang communicate polarity information between neighboring cells is poorly understood. The atypical cadherin, Flamingo (Fmi), is implicated in this process, yet whether Fmi acts permissively as a scaffold or instructively as a signal is unclear. Here, we provide evidence that Fmi functions instructively to mediate Fz-Vang intercellular signal relay, recruiting Fz and Vang to opposite sides of cell boundaries. We propose that two functional forms of Fmi, one of which is induced by and physically interacts with Fz, bind each other to create cadherin homodimers that signal bidirectionally and asymmetrically, instructing unequal responses in adjacent cell membranes to establish molecular asymmetry.

  1. Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells

    PubMed Central

    Souilhol, Céline; Gonneau, Christèle; Lendinez, Javier G.; Batsivari, Antoniana; Rybtsov, Stanislav; Wilson, Heather; Morgado-Palacin, Lucia; Hills, David; Taoudi, Samir; Antonchuk, Jennifer; Zhao, Suling; Medvinsky, Alexander

    2016-01-01

    During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta–gonad–mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso–ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs. PMID:26952187

  2. Antidepressant effects of sleep deprivation require astrocyte-dependent adenosine mediated signaling

    PubMed Central

    Hines, D J; Schmitt, L I; Hines, R M; Moss, S J; Haydon, P G

    2013-01-01

    Major depressive disorder is a debilitating condition with a lifetime risk of ten percent. Most treatments take several weeks to achieve clinical efficacy, limiting the ability to bring instant relief needed in psychiatric emergencies. One intervention that rapidly alleviates depressive symptoms is sleep deprivation; however, its mechanism of action is unknown. Astrocytes regulate responses to sleep deprivation, raising the possibility that glial signaling mediates antidepressive-like actions of sleep deprivation. Here, we found that astrocytic signaling to adenosine (A1) receptors was required for the robust reduction of depressive-like behaviors following 12 hours of sleep deprivation. As sleep deprivation activates synaptic A1 receptors, we mimicked the effect of sleep deprivation on depression phenotypes by administration of the A1 agonist CCPA. These results provide the first mechanistic insight into how sleep deprivation impacts mood, and provide a novel pathway for rapid antidepressant development by modulation of glial signaling in the brain. PMID:23321809

  3. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    SciTech Connect

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression.

  4. BMP Signaling Mediates Effects of Exercise on Hippocampal Neurogenesis and Cognition in Mice

    PubMed Central

    Gobeske, Kevin T.; Das, Sunit; Bonaguidi, Michael A.; Weiss, Craig; Radulovic, Jelena; Disterhoft, John F.; Kessler, John A.

    2009-01-01

    Exposure to exercise or to environmental enrichment increases the generation of new neurons in the adult hippocampus and promotes certain kinds of learning and memory. While the precise role of neurogenesis in cognition has been debated intensely, comparatively few studies have addressed the mechanisms linking environmental exposures to cellular and behavioral outcomes. Here we show that bone morphogenetic protein (BMP) signaling mediates the effects of exercise on neurogenesis and cognition in the adult hippocampus. Elective exercise reduces levels of hippocampal BMP signaling before and during its promotion of neurogenesis and learning. Transgenic mice with decreased BMP signaling or wild type mice infused with a BMP inhibitor both exhibit remarkable gains in hippocampal cognitive performance and neurogenesis, mirroring the effects of exercise. Conversely, transgenic mice with increased BMP signaling have diminished hippocampal neurogenesis and impaired cognition. Exercise exposure does not rescue these deficits, suggesting that reduced BMP signaling is required for environmental effects on neurogenesis and learning. Together, these observations show that BMP signaling is a fundamental mechanism linking environmental exposure with changes in cognitive function and cellular properties in the hippocampus. PMID:19841742

  5. α1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice.

    PubMed

    Ding, Fengfei; O'Donnell, John; Thrane, Alexander S; Zeppenfeld, Douglas; Kang, Hongyi; Xie, Lulu; Wang, Fushun; Nedergaard, Maiken

    2013-12-01

    Astrocyte Ca2+ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca2+ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca2+ signals by activation of astrocytic α1-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca2+ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α1-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca2+ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca2+ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca2+ signaling being triggered by the α1-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity.

  6. The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1

    PubMed Central

    2016-01-01

    Abstract Pulmonary arterial hypertension (PAH) is a devastating vasculopathy that predominates in women and has been associated with dysregulated estrogen and serotonin signaling. Overexpression of the serotonin transporter (SERT+) in mice results in an estrogen-dependent development of pulmonary hypertension (PH). Estrogen metabolism by cytochrome P450 1B1 (CYP1B1) contributes to the pathogenesis of PAH, and serotonin can increase CYP1B1 expression in human pulmonary arterial smooth muscle cells (hPASMCs). We hypothesized that an increase in intracellular serotonin via increased SERT expression may dysregulate estrogen metabolism via CYP1B1 to facilitate PAH. Consistent with this hypothesis, we found elevated lung CYP1B1 protein expression in female SERT+ mice accompanied by PH, which was attenuated by the CYP1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS). Lungs from female SERT+ mice demonstrated an increase in oxidative stress that was marked by the expression of 8-hydroxyguanosine; however, this was unaffected by CYP1B1 inhibition. SERT expression was increased in monocrotaline-induced PH in female rats; however, TMS did not reverse PH in monocrotaline-treated rats but prolonged survival. Stimulation of hPASMCs with the CYP1B1 metabolite 16α-hydroxyestrone increased cellular proliferation, which was attenuated by an inhibitor (MPP) of estrogen receptor alpha (ERα) and a specific ERα antibody. Thus, increased intracellular serotonin caused by increased SERT expression may contribute to PAH pathobiology by dysregulation of estrogen metabolic pathways via increased CYP1B1 activity. This promotes PASMC proliferation by the formation of pathogenic metabolites of estrogen that mediate their effects via ERα. Our studies indicate that targeting this pathway in PAH may provide a promising antiproliferative therapeutic strategy. PMID:27162617

  7. Inhibition of M current in sensory neurons by exogenous proteases: a signaling pathway mediating inflammatory nociception.

    PubMed

    Linley, John E; Rose, Kirstin; Patil, Mayur; Robertson, Brian; Akopian, Armen N; Gamper, Nikita

    2008-10-29

    Inflammatory pain is thought to be mediated in part through the action of inflammatory mediators on membrane receptors of peripheral nerve terminals, however, the downstream signaling events which lead to pain are poorly understood. In this study we investigated the nociceptive pathways induced by activation of protease-activated receptor 2 (PAR-2) in damage-sensing (nociceptive) neurons from rat dorsal root ganglion (DRG). We found that activation of PAR-2 in these cells strongly inhibited M-type potassium currents (conducted by Kv7 potassium channels). Such inhibition caused depolarization of the neuronal resting membrane potential leading, ultimately, to nociception. Consistent with this mechanism, injection of the specific M channel blocker XE991 into rat paw induced nociception in a concentration-dependent manner. Injection of a PAR-2 agonist peptide also induced nociception but coinjection of XE991 and the PAR-2 agonist did not result in summation of nociception, suggesting that the action of both agents may share a similar mechanism. We also studied the signaling pathway of M current inhibition by PAR-2 using patch-clamp and fluorescence imaging of DRG neurons. These experiments revealed that the PAR-2 effect was mediated by phospholipase C (PLC). Further experiments demonstrated that M current inhibition required concurrent rises in cytosolic Ca(2+) concentration and depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We propose that PLC- and Ca(2+)/PIP(2)-mediated inhibition of M current in sensory neurons may represent one of the general mechanisms underlying pain produced by inflammatory mediators, and may therefore open up a new therapeutic window for treatment of this major clinical problem. PMID:18971466

  8. Control of metabolic homeostasis by stress signaling is mediated by the lipocalin NLaz.

    PubMed

    Hull-Thompson, Julie; Muffat, Julien; Sanchez, Diego; Walker, David W; Benzer, Seymour; Ganfornina, Maria D; Jasper, Heinrich

    2009-04-01

    Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways--such as Jun-N-terminal Kinase (JNK) signaling--repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. Here, we identify a new regulatory mechanism by which JNK influences metabolism systemically. We show that JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan--phenotypes that are consistent with reduced IIS activity. Accordingly, we find that NLaz represses IIS activity in larvae and adult flies. Our results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis.

  9. A Model for Carrier-Mediated Biological Signal Transduction Based on Equilibrium Ligand Binding Theory.

    PubMed

    Martini, Johannes W R; Schlather, Martin; Schütz, Stefan

    2016-05-01

    Different variants of a mathematical model for carrier-mediated signal transduction are introduced with focus on the odor dose-electrophysiological response curve of insect olfaction. The latter offers a unique opportunity to observe experimentally the effect of an alteration in the carrier molecule composition on the signal molecule-dependent response curve. Our work highlights the role of involved carrier molecules, which have largely been ignored in mathematical models for response curves in the past. The resulting model explains how the involvement of more than one carrier molecule in signal molecule transport can cause dose-response curves as observed in experiments, without the need of more than one receptor per neuron. In particular, the model has the following features: (1) An extended sensitivity range of neuronal response is implemented by a system consisting of only one receptor but several carrier molecules with different affinities for the signal molecule. (2) Given that the sensitivity range is extended by the involvement of different carrier molecules, the model implies that a strong difference in the expression levels of the carrier molecules is absolutely essential for wide range responses. (3) Complex changes in dose-response curves which can be observed when the expression levels of carrier molecules are altered experimentally can be explained by interactions between different carrier molecules. The principles we demonstrate here for electrophysiological responses can also be applied to any other carrier-mediated biological signal transduction process. The presented concept provides a framework for modeling and statistical analysis of signal transduction processes if sufficient information on the underlying biology is available.

  10. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  11. Aldo-Keto Reductases 1B in Endocrinology and Metabolism

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2012-01-01

    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers. PMID:22876234

  12. Aldo-Keto Reductases 1B in Endocrinology and Metabolism.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2012-01-01

    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers.

  13. Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells

    PubMed Central

    Lee, S-J; Jung, Y H; Oh, S Y; Jang, K K; Lee, H S; Choi, S H; Han, H J

    2015-01-01

    Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-β-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells. PMID:26086960

  14. Autocrine TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in breast cells.

    PubMed

    Yang, L; Yang, J; Venkateswarlu, S; Ko, T; Brattain, M G

    2001-09-01

    In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also

  15. In Silico Approach for SAR Analysis of the Predicted Model of DEPDC1B: A Novel Target for Oral Cancer.

    PubMed

    Ahuja, Palak; Singh, Kailash

    2016-01-01

    With the incidence rate of oral carcinogenesis increasing in the Southeast-Asian countries, due to increase in the consumption of tobacco and betel quid as well as infection from human papillomavirus, specifically type 16, it becomes crucial to predict the transition of premalignant lesion to cancerous tissue at an initial stage in order to control the process of oncogenesis. DEPDC1B, downregulated in the presence of E2 protein, was recently found to be overexpressed in oral cancer, which can possibly be explained by the disruption of the E2 open reading frame upon the integration of viral genome into the host genome. DEPDC1B mediates its effect by directly interacting with Rac1 protein, which is known to regulate important cell signaling pathways. Therefore, DEPDC1B can be a potential biomarker as well as a therapeutic target for diagnosing and curing the disease. However, the lack of 3D model of the structure makes the utilization of DEPDC1B as a therapeutic target difficult. The present study focuses on the prediction of a suitable 3D model of the protein as well as the analysis of protein-protein interaction between DEPDC1B and Rac1 protein using PatchDock web server along with the identification of allosteric or regulatory sites of DEPDC1B. PMID:27034663

  16. Validation of signalling pathways: Case study of the p16-mediated pathway.

    PubMed

    Akçay, Nimet İlke; Bashirov, Rza; Tüzmen, Şükrü

    2015-04-01

    p16 is recognized as a tumor suppressor gene due to the prevalence of its genetic inactivation in all types of human cancers. Additionally, p16 gene plays a critical role in controlling aging, regulating cellular senescence, detection and maintenance of DNA damage. The molecular mechanism behind these events involves p16-mediated signaling pathway (or p16- Rb pathway), the focus of our study. Understanding functional dependence between dynamic behavior of biological components involved in the p16-mediated pathway and aforesaid molecular-level events might suggest possible implications in the diagnosis, prognosis and treatment of human cancer. In the present work, we employ reverse-engineering approach to construct the most detailed computational model of p16-mediated pathway in higher eukaryotes. We implement experimental data from the literature to validate the model, and under various assumptions predict the dynamic behavior of p16 and other biological components by interpreting the simulation results. The quantitative model of p16-mediated pathway is created in a systematic manner in terms of Petri net technologies.

  17. Control of Metabolic Homeostasis by Stress Signaling Is Mediated by the Lipocalin NLaz

    PubMed Central

    Sanchez, Diego; Walker, David W.; Benzer, Seymour; Ganfornina, Maria D.; Jasper, Heinrich

    2009-01-01

    Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways—such as Jun-N-terminal Kinase (JNK) signaling—repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. Here, we identify a new regulatory mechanism by which JNK influences metabolism systemically. We show that JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan—phenotypes that are consistent with reduced IIS activity. Accordingly, we find that NLaz represses IIS activity in larvae and adult flies. Our results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis. PMID:19390610

  18. Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

    SciTech Connect

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad

    2013-11-01

    Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

  19. A pain-mediated neural signal induces relapse in murine autoimmune encephalomyelitis, a multiple sclerosis model

    PubMed Central

    Arima, Yasunobu; Kamimura, Daisuke; Atsumi, Toru; Harada, Masaya; Kawamoto, Tadafumi; Nishikawa, Naoki; Stofkova, Andrea; Ohki, Takuto; Higuchi, Kotaro; Morimoto, Yuji; Wieghofer, Peter; Okada, Yuka; Mori, Yuki; Sakoda, Saburo; Saika, Shizuya; Yoshioka, Yoshichika; Komuro, Issei; Yamashita, Toshihide; Hirano, Toshio; Prinz, Marco; Murakami, Masaaki

    2015-01-01

    Although pain is a common symptom of various diseases and disorders, its contribution to disease pathogenesis is not well understood. Here we show using murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), that pain induces EAE relapse. Mechanistic analysis showed that pain induction activates a sensory-sympathetic signal followed by a chemokine-mediated accumulation of MHC class II+CD11b+ cells that showed antigen-presentation activity at specific ventral vessels in the fifth lumbar cord of EAE-recovered mice. Following this accumulation, various immune cells including pathogenic CD4+ T cells recruited in the spinal cord in a manner dependent on a local chemokine inducer in endothelial cells, resulting in EAE relapse. Our results demonstrate that a pain-mediated neural signal can be transformed into an inflammation reaction at specific vessels to induce disease relapse, thus making this signal a potential therapeutic target. DOI: http://dx.doi.org/10.7554/eLife.08733.001 PMID:26193120

  20. Curcumin Rescues Diabetic Renal Fibrosis by Targeting Superoxide-Mediated Wnt Signaling Pathways.

    PubMed

    Ho, Cheng; Hsu, Yung-Chien; Lei, Chen-Chou; Mau, Shu-Ching; Shih, Ya-Hsueh; Lin, Chun-Liang

    2016-03-01

    The purposes of this study were to investigate whether curcumin can weaken diabetic nephropathy by modulating both oxidative stress and renal injury from Wnt signaling mediation. Wnt5a/β-catenin depression and induction of superoxide synthesis are associated with high glucose (HG) induced transforming growth factor (TGF)-β1 and fibronectin expression in mesangial cells. Curcumin resumes HG depression of Wnt/β-catenin signaling and alleviates HG induction of superoxide, TGF-β1 and fibronectin expression in renal mesangial cell. Exogenous curcumin alleviated urinary total proteinuria and serum superoxide level in diabetic rats. Based on laser-captured microdissection for quantitative real-time polymerase chain reaction, it was found that diabetes significantly increased TGF-β1 and fibronectin expression in line with depressed Wnt5a expression. Curcumin treatment reduced the TGF-β1 and fibronectin activation and the inhibiting effect of diabetes on Wnt5a/β-catenin expression in renal glomeruli. Immunohistochemistry showed that curcumin treatment significantly reduced 8-hydroxy-2'-deoxyguanosine, TGF-β1 and fibronectin, and was in line with the restoration of the suppressed Wnt5a expression immunoreactivities in glomeruli of diabetic rats. Curcumin alleviated extracellular matrix accumulation in diabetic nephropathy by not only preventing the diabetes-mediated superoxide synthesis but also resuming downregulation of Wnt/β-catenin signaling. These findings suggest that regulation of Wnt activity by curcumin is a feasible alternative strategy to rescue diabetic renal injury.

  1. Interplay between Dioxin-Mediated Signaling and Circadian Clock: A Possible Determinant in Metabolic Homeostasis

    PubMed Central

    Wang, Chun; Zhang, Zhi-Ming; Xu, Can-Xin; Tischkau, Shelley A.

    2014-01-01

    The rotation of the earth on its axis creates the environment of a 24 h solar day, which organisms on earth have used to their evolutionary advantage by integrating this timing information into their genetic make-up in the form of a circadian clock. This intrinsic molecular clock is pivotal for maintenance of synchronized homeostasis between the individual organism and the external environment to allow coordinated rhythmic physiological and behavioral function. Aryl hydrocarbon receptor (AhR) is a master regulator of dioxin-mediated toxic effects, and is, therefore, critical in maintaining adaptive responses through regulating the expression of phase I/II drug metabolism enzymes. AhR expression is robustly rhythmic, and physiological cross-talk between AhR signaling and circadian rhythms has been established. Increasing evidence raises a compelling argument that disruption of endogenous circadian rhythms contributes to the development of disease, including sleep disorders, metabolic disorders and cancers. Similarly, exposure to environmental pollutants through air, water and food, is increasingly cited as contributory to these same problems. Thus, a better understanding of interactions between AhR signaling and the circadian clock regulatory network can provide critical new insights into environmentally regulated disease processes. This review highlights recent advances in the understanding of the reciprocal interactions between dioxin-mediated AhR signaling and the circadian clock including how these pathways relate to health and disease, with emphasis on the control of metabolic function. PMID:24987953

  2. Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis.

    PubMed

    Huynh-Do, Uyen; Vindis, Cécile; Liu, Hua; Cerretti, Douglas Pat; McGrew, Jeffrey T; Enriquez, Miriam; Chen, Jin; Daniel, Thomas O

    2002-08-01

    Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (alpha(v)beta(3) and alpha(5)beta(1)) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1DeltaCy) or a deletion of four C-terminal amino acids (ephrin-B1DeltaPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1DeltaCy and ephrin-B1DeltaPDZbd mutants were inactive. Thus ephrin-B1 transduces 'outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration. PMID:12118063

  3. Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

    PubMed

    Venugopal, Srivathsa C; Jeong, Rae-Dong; Mandal, Mihir K; Zhu, Shifeng; Chandra-Shekara, A C; Xia, Ye; Hersh, Matthew; Stromberg, Arnold J; Navarre, DuRoy; Kachroo, Aardra; Kachroo, Pradeep

    2009-07-01

    Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

  4. Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy

    PubMed Central

    Bernt, Alexander; Rangrez, Ashraf Y.; Eden, Matthias; Jungmann, Andreas; Katz, Sylvia; Rohr, Claudia; Müller, Oliver J.; Katus, Hugo A.; Sossalla, Samuel T.; Williams, Tatjana; Ritter, Oliver; Frank, Derk; Frey, Norbert

    2016-01-01

    The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~107 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway. PMID:27767176

  5. Identification of signaling pathways regulating primary cilium length and flow-mediated adaptation.

    PubMed

    Besschetnova, Tatiana Y; Kolpakova-Hart, Elona; Guan, Yinghua; Zhou, Jing; Olsen, Bjorn R; Shah, Jagesh V

    2010-01-26

    The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

  6. Curcumin Rescues Diabetic Renal Fibrosis by Targeting Superoxide-Mediated Wnt Signaling Pathways.

    PubMed

    Ho, Cheng; Hsu, Yung-Chien; Lei, Chen-Chou; Mau, Shu-Ching; Shih, Ya-Hsueh; Lin, Chun-Liang

    2016-03-01

    The purposes of this study were to investigate whether curcumin can weaken diabetic nephropathy by modulating both oxidative stress and renal injury from Wnt signaling mediation. Wnt5a/β-catenin depression and induction of superoxide synthesis are associated with high glucose (HG) induced transforming growth factor (TGF)-β1 and fibronectin expression in mesangial cells. Curcumin resumes HG depression of Wnt/β-catenin signaling and alleviates HG induction of superoxide, TGF-β1 and fibronectin expression in renal mesangial cell. Exogenous curcumin alleviated urinary total proteinuria and serum superoxide level in diabetic rats. Based on laser-captured microdissection for quantitative real-time polymerase chain reaction, it was found that diabetes significantly increased TGF-β1 and fibronectin expression in line with depressed Wnt5a expression. Curcumin treatment reduced the TGF-β1 and fibronectin activation and the inhibiting effect of diabetes on Wnt5a/β-catenin expression in renal glomeruli. Immunohistochemistry showed that curcumin treatment significantly reduced 8-hydroxy-2'-deoxyguanosine, TGF-β1 and fibronectin, and was in line with the restoration of the suppressed Wnt5a expression immunoreactivities in glomeruli of diabetic rats. Curcumin alleviated extracellular matrix accumulation in diabetic nephropathy by not only preventing the diabetes-mediated superoxide synthesis but also resuming downregulation of Wnt/β-catenin signaling. These findings suggest that regulation of Wnt activity by curcumin is a feasible alternative strategy to rescue diabetic renal injury. PMID:26992258

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

    PubMed

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

    2015-09-01

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

  8. Activation of Notch1 signaling is required for β-catenin–mediated human primary melanoma progression

    PubMed Central

    Balint, Klara; Xiao, Min; Pinnix, Chelsea C.; Soma, Akinobu; Veres, Imre; Juhasz, Istvan; Brown, Eric J.; Capobianco, Anthony J.; Herlyn, Meenhard; Liu, Zhao-Jun

    2005-01-01

    Notch is a highly conserved transmembrane receptor that determines cell fate. Notch signaling denotes cleavage of the Notch intracellular domain, its translocation to the nucleus, and subsequent activation of target gene transcription. Involvement of Notch signaling in several cancers is well known, but its role in melanoma remains poorly characterized. Here we show that the Notch1 pathway is activated in human melanoma. Blocking Notch signaling suppressed whereas constitutive activation of the Notch1 pathway enhanced primary melanoma cell growth both in vitro and in vivo yet had little effect on metastatic melanoma cells. Activation of Notch1 signaling enabled primary melanoma cells to gain metastatic capability. Furthermore, the oncogenic effect of Notch1 on primary melanoma cells was mediated by β-catenin, which was upregulated following Notch1 activation. Inhibiting β-catenin expression reversed Notch1-enhanced tumor growth and metastasis. Our data therefore suggest a β-catenin–dependent, stage-specific role for Notch1 signaling in promoting the progression of primary melanoma. PMID:16239965

  9. CD38-mediated Ca(2+) signaling contributes to glucagon-induced hepatic gluconeogenesis.

    PubMed

    Rah, So-Young; Kim, Uh-Hyun

    2015-06-03

    CD38 is a multifunctional enzyme for the synthesis of Ca(2+) second messengers. Glucagon promotes hepatic glucose production through Ca(2+) signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca(2+) increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca(2+) increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca(2+) signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38(-/-) mice. Furthermore, in the fasting condition, CD38(-/-) mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca(2+) signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes.

  10. P2y Receptor-Mediated Angiogenesis via Vascular Endothelial Growth Factor Receptor 2 Signaling

    PubMed Central

    Rumjahn, Sharif M.; Baldwin, Karla A; Buxton, Iain L. O.

    2011-01-01

    Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 μM ATP and 10 μM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 μM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis. PMID:18605230

  11. FoxK mediates TGF-β signalling during midgut differentiation in flies

    PubMed Central

    Casas-Tinto, Sergio; Gomez-Velazquez, Melisa; Granadino, Begoña; Fernandez-Funez, Pedro

    2008-01-01

    Inductive signals across germ layers are important for the development of the endoderm in vertebrates and invertebrates (Tam, P.P., M. Kanai-Azuma, and Y. Kanai. 2003. Curr. Opin. Genet. Dev. 13:393–400; Nakagoshi, H. 2005. Dev. Growth Differ. 47:383–392). In flies, the visceral mesoderm secretes signaling molecules that diffuse into the underlying midgut endoderm, where conserved signaling cascades activate the Hox gene labial, which is important for the differentiation of copper cells (Bienz, M. 1997. Curr. Opin. Genet. Dev. 7:683–688). We present here a Drosophila melanogaster gene of the Fox family of transcription factors, FoxK, that mediates transforming growth factor β (TGF-β) signaling in the embryonic midgut endoderm. FoxK mutant embryos fail to generate midgut constrictions and lack Labial in the endoderm. Our observations suggest that TGF-β signaling directly regulates FoxK through functional Smad/Mad-binding sites, whereas FoxK, in turn, regulates labial expression. We also describe a new cooperative activity of the transcription factors FoxK and Dfos/AP-1 that regulates labial expression in the midgut endoderm. This regulatory activity does not require direct labial activation by the TGF-β effector Mad. Thus, we propose that the combined activity of the TGF-β target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm. PMID:19075113

  12. Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

    PubMed Central

    Miller, Evan W.; Dickinson, Bryan C.; Chang, Christopher J.

    2010-01-01

    Hydrogen peroxide (H2O2) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H2O2 to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H2O2 into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H2O2, directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H2O2 specifically through membranes in mammalian cells. Moreover, we show that intracellular H2O2 accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Nox-derived H2O2 signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H2O2 can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions. PMID:20724658

  13. CD38-mediated Ca2+ signaling contributes to glucagon-induced hepatic gluconeogenesis

    PubMed Central

    Rah, So-Young; Kim, Uh-Hyun

    2015-01-01

    CD38 is a multifunctional enzyme for the synthesis of Ca2+ second messengers. Glucagon promotes hepatic glucose production through Ca2+ signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca2+ increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca2+ increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca2+ signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38−/− mice. Furthermore, in the fasting condition, CD38−/− mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca2+ signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes. PMID:26038839

  14. Human autoreactive T cells recognize CD1b and phospholipids

    PubMed Central

    Van Rhijn, Ildiko; van Berlo, Twan; Hilmenyuk, Tamara; Cheng, Tan-Yun; Wolf, Benjamin J.; Tatituri, Raju V. V.; Uldrich, Adam P.; Napolitani, Giorgio; Cerundolo, Vincenzo; Altman, John D.; Willemsen, Peter; Huang, Shouxiong; Rossjohn, Jamie; Besra, Gurdyal S.; Brenner, Michael B.; Godfrey, Dale I.; Moody, D. Branch

    2016-01-01

    In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids. PMID:26621732

  15. Copper(i)-Y zeolite catalyzed N-sulfonylketenimine mediated annulation of hydroxynaphthoquinones: syntheses of naphtho[2,1-b]furan-2,5-diones and benzo[de]chromene-2,6-diones.

    PubMed

    Ramanathan, Devenderan; Namitharan, Kayambu; Pitchumani, Kasi

    2016-06-28

    An efficient one pot synthesis for the construction of novel naphtho[2,1-b]furan-2,5-diones and benzo[de]chromene-2,6-diones using copper(i)-Y zeolite catalyzed reaction of N-sulfonylketenimine with 2-hydroxy-1,4-naphthoquinone and 5-hydroxy-1,4-naphthoquinone followed by the elimination of p-toluenesulfonamide is reported. The intermediate N-sulfonylketenimine, generated by (3+2) cycloaddition/ring-opening reaction/retro-Wolff rearrangement, cascade, and annulation, promotes the reaction involving the inter- and intramolecular nucleophilic addition/dehydration followed by hydrolysis and elimination of p-toluenesulfonamide to afford the target products in good yield. PMID:27305854

  16. Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture

    PubMed Central

    McDonald, John L.; Cripps, Allan W.; Smith, Peter K.

    2015-01-01

    Acupuncture has been used for millennia to treat allergic diseases including both intermittent rhinitis and persistent rhinitis. Besides the research on the efficacy and safety of acupuncture treatment for allergic rhinitis, research has also investigated how acupuncture might modulate immune function to exert anti-inflammatory effects. A proposed model has previously hypothesized that acupuncture might downregulate proinflammatory neuropeptides, proinflammatory cytokines, and neurotrophins, modulating transient receptor potential vallinoid (TRPV1), a G-protein coupled receptor which plays a central role in allergic rhinitis. Recent research has been largely supportive of this model. New advances in research include the discovery of a novel cholinergic anti-inflammatory pathway activated by acupuncture. A chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues, in response to acupuncture, has also been demonstrated for the first time. Further research on the complex cross talk between receptors during inflammation is also helping to elucidate the mediators and signalling pathways activated by acupuncture. PMID:26339274

  17. Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint

    PubMed Central

    London, Nitobe; Biggins, Sue

    2014-01-01

    The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint. PMID:24402315

  18. Ca2+ channels as integrators of G protein-mediated signaling in neurons.

    PubMed

    Strock, Jesse; Diversé-Pierluissi, María A

    2004-11-01

    The observations from Dunlap and Fischbach that transmitter-mediated shortening of the duration of action potentials could be caused by a decrease in calcium conductance led to numerous studies of the mechanisms of modulation of voltage-dependent calcium channels. Calcium channels are well known targets for inhibition by receptor-G protein pathways, and multiple forms of inhibition have been described. Inhibition of Ca(2+) channels can be mediated by G protein betagamma-subunits or by kinases, such as protein kinase C and tyrosine kinases. In the last few years, it has been shown that integration of G protein signaling can take place at the level of the calcium channel by regulation of the interaction of the channel pore-forming subunit with different cellular proteins.

  19. Gamma-ray signals from dark matter annihilation via charged mediators

    NASA Astrophysics Data System (ADS)

    Kumar, Jason; Sandick, Pearl; Teng, Fei; Yamamoto, Takahiro

    2016-07-01

    We consider a simplified model in which Majorana fermion dark matter annihilates to charged fermions through the exchange of charged mediators. We consider the gamma-ray signals arising from the processes X X →f ¯ f γ , γ γ , and γ Z in the most general case, including nontrivial fermion mass and nontrivial left-right mixing and the C P -violating phase for the charged mediators. In particular, we find the most general spectrum for internal bremsstrahlung, which interpolates between the regimes dominated by virtual internal bremsstrahlung and by final state radiation. We also examine the variation in the ratio σ (γ γ )/σ (γ Z ) and the helicity asymmetry in the X X →γ γ process, each as a function of the mixing angle and C P -violating phase. As an application, we apply these results to searches for a class of minimal supersymmetric Standard Model models.

  20. Cloning/Characterization of the Canine Organic Anion Transporting Polypeptide 1b4 (Oatp1b4) and Classification of the Canine OATP/SLCO Members

    PubMed Central

    Gui, Chunshan; Hagenbuch, Bruno

    2010-01-01

    The human liver specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076 bp, encoding a 692-amino acid protein with a molecular mass of ~85 kDa. The Oatp1b4 gene is approximately 61 kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine2,5]enkephalin (DPDPE), estradiol-17β-glucuronide (E17βG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17βG and estrone-3-sulfate transports were monophasic with Km values of 5 ± 1 μM and 33 ± 4 μM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members. PMID:20079461

  1. Unmasking of a Protective TNFR1 Mediated Signal in the Collagen Arthritis Model

    PubMed Central

    Williams-Skipp, Cheryll; Raman, Thiagarajan; Valuck, Robert J.; Watkins, Herschel; Palmer, Brent E.; Scheinman, Robert I.

    2009-01-01

    OBJECTIVE: TNFR1 plays a major role in rheumatoid arthritis (RA). Here we explore the relative importance of TNFR1 signaling in the hematopoietic tissue compartment for disease progression. METHODS: DBA/1 mice were lethally irradiated and rescued with bone marrow derived from either DBA/1 or TNFR1−/− animals. The mice were then input into the collagen induced arthritis (CIA) model and disease progression characterized. RESULTS: Surprisingly, TNFR1−/− transplant mice input into the CIA model develop increased disease as compared to controls. This could not be attributed to either an increased primary response to collagen or to the contribution of a non-DBA genetic background. Histological markers of advanced disease were evident in TNFR1−/− transplant mice shortly after initiation of the immune response to collagen and long before clinical evidence of disease. Serum TNFα was undetectable while serum IL-12p40 levels were increased in TNFR1−/− transplant mice at the end point of the study. CONCLUSION: These data raise the intriguing possibility of the existence of an anti-inflammatory TNFR1 mediated circuit in the hematopoietic compartment. This circuit bears a resemblance to emerging data delineating a switch in TNFα function observed in the resolution of bacterial infections. These data suggest that TNFR1 mediated signals in the radio-resistant tissues contributes to disease progression while TNFR1 mediated signals in the radio-sensitive tissues can contribute to protection from disease. We thus put forward the hypothesis that the degree of responce to TNFα blockade in RA is dependent, in part, on the relative genetic strengths of these two pathways. PMID:19180511

  2. Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish

    PubMed Central

    Thakur, Prakash C.; Davison, Jon M.; Stuckenholz, Carsten; Lu, Lili; Bahary, Nathan

    2014-01-01

    Dysregulated phosphatidylinositol (PI) signaling has been implicated in human gastrointestinal (GI) malignancies and inflammatory states, underlining the need to study pathophysiological roles of PI in an in vivo genetic model. Here, we study the significance of PI in GI pathophysiology using the zebrafish mutant cdipthi559, which lacks PI synthesis, and unravel a crucial role of PI in intestinal mucosal integrity and inflammation. The cdipthi559 mutants exhibit abnormal villous architecture and disorganized proliferation of intestinal epithelial cells (IECs), with pathologies reminiscent of inflammatory bowel disease (IBD), including apoptosis of goblet cells, abnormal mucosecretion, bacterial overgrowth and leukocyte infiltration. The mutant IECs exhibit vacuolation, microvillus atrophy and impaired proliferation. The cdipthi559 gene expression profile shows enrichment of acute phase response signaling, and the endoplasmic reticulum (ER) stress factors hspa5 and xbp1 are robustly activated in the mutant GI tissue. Temporal electron micrographic analyses reveal that PI-deficient IECs undergo sequential ER-Golgi disruption, mitochondrial depletion, macroautophagy and cell death, consistent with chronic ER-stress-mediated cytopathology. Furthermore, pharmacological induction of ER stress by inhibiting protein glycosylation or PI synthase inhibition in leukocyte-specific reporter lines replicates the cdipthi559 inflammatory phenotype, suggesting a fundamental role of PI metabolism and ER stress in mucosal inflammation. Antibiotics and anti-inflammatory drugs resolved the inflammation, but not the autophagic necroapoptosis of IECs, suggesting that bacterial overgrowth can exacerbate ER stress pathology, whereas persistent ER stress is sufficient to trigger inflammation. Interestingly, the intestinal phenotype was partially alleviated by chemical chaperones, suggesting their therapeutic potential. Using zebrafish genetic and pharmacological models, this study

  3. Gpr177-mediated Wnt Signaling Is Required for Secondary Palate Development.

    PubMed

    Liu, Y; Wang, M; Zhao, W; Yuan, X; Yang, X; Li, Y; Qiu, M; Zhu, X-J; Zhang, Z

    2015-07-01

    Cleft palate represents one of the major congenital birth defects in humans. Despite the essential roles of ectodermal canonical Wnt and mesenchymal Wnt signaling in the secondary palate development, the function of mesenchymal canonical Wnt activity in secondary palate development remains elusive. Here we show that Gpr177, a highly conserved transmembrane protein essential for Wnt trafficking, is required for secondary palate development. Gpr177 is expressed in both epithelium and mesenchyme of palatal shelves during mouse development. Wnt1(Cre)-mediated deletion of Gpr177 in craniofacial neural crest cells leads to a complete cleft secondary palate, which is formed mainly due to aberrant cell proliferation and increased cell death in palatal shelves. By BATGAL staining, we reveal an intense canonical Wnt activity in the anterior palate mesenchyme of E12.5 wild-type embryos but not in Gpr177(Wnt1-Cre) embryos, suggesting that mesenchymal canonical Wnt signaling activated by Gpr177-mediated mesenchymal Wnts is critical for secondary palate development. Moreover, phosphorylation of JNK and c-Jun is impaired in the Gpr177(Wnt1-Cre) palate and is restored by implantation of Wnt5a-soaked beads in the in vitro palate explants, suggesting that Gpr177 probably regulates palate development via the Wnt5a-mediated noncanonical Wnt pathway in which c-Jun and JNK are involved. Importantly, certain cellular processes and the altered gene expression in palates lacking Gpr177 are distinct from that of the Wnt5a mutant, further demonstrating involvement of other mesenchymal Wnts in the process of palate development. Together, these results suggest that mesenchymal Gpr177 is required for secondary palate development by regulating and integrating mesenchymal canonical and noncanonical Wnt signals.

  4. Mechanical strain promotes osteoblastic differentiation through integrin-β1-mediated β-catenin signaling.

    PubMed

    Yan, Yuxian; Sun, Haoyang; Gong, Yuanwei; Yan, Zhixiong; Zhang, Xizheng; Guo, Yong; Wang, Yang

    2016-08-01

    As integrins are mechanoresponsive, there exists an intimate relationship between integrins and mechanical strain. Integrin-β1 mediates the impact of mechanical strain on bone. Mechanical strain induces bone formation through the activation of β-catenin pathways, which suggests that integrin-β1 mediates β-catenin signaling in osteoblasts in response to mechanical strain. In the present study, we examined the role of integrin-β1 in Wnt/β-catenin signal transduction in mechanically strained osteoblasts. MC3T3-E1 osteoblastic cells were transfected with integrin-β1 small interfering RNA (si-Itgβ1), and exposed to mechanical tensile strain of 2,500 microstrain (µε) using a four-point bending device. The mechanical strain enhanced the mRNA expression of integrin-β1, the protein levels of phosphorylated (p-) glycogen synthase kinase-3β (GSK‑3β) and β-catenin, simultaneously increased the mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), the protein levels of bone morphogenetic protein (BMP)-2 and -4 and enhanced the alkaline phosphatase (ALP) activity of the ME3T3-E1 cells. The elevations were inhibited by si-Itgβ1. Additionally, the mechanical strain induced the nuclear translocation of β-catenin into the nucleus, which was also inhibited by si-Itgβ1. These findings indicated that mechanical strain promoted osteoblastic differentiation through integrin‑β1‑mediated β-catenin signaling.

  5. Alternative erythropoietin-mediated signaling prevents secondary microvascular thrombosis and inflammation within cutaneous burns.

    PubMed

    Bohr, Stefan; Patel, Suraj J; Shen, Keyue; Vitalo, Antonia G; Brines, Michael; Cerami, Anthony; Berthiaume, Francois; Yarmush, Martin L

    2013-02-26

    Alternate erythropoietin (EPO)-mediated signaling via the heteromeric receptor composed of the EPO receptor and the β-common receptor (CD131) exerts the tissue-protective actions of EPO in various types of injuries. Herein we investigated the effects of the EPO derivative helix beta surface peptide (synonym: ARA290), which specifically triggers alternate EPO-mediated signaling, but does not bind the erythropoietic EPO receptor homodimer, on the progression of secondary tissue damage following cutaneous burns. For this purpose, a deep partial thickness cutaneous burn injury was applied on the back of mice, followed by systemic administration of vehicle or ARA290 at 1, 12, and 24 h postburn. With vehicle-only treatment, wounds exhibited secondary microvascular thrombosis within 24 h postburn, and subsequent necrosis of the surrounding tissue, thus converting to a full-thickness injury within 48 h. On the other hand, when ARA290 was systemically administered, patency of the microvasculature was maintained. Furthermore, ARA290 mitigated the innate inflammatory response, most notably tumor necrosis factor-alpha-mediated signaling. These findings correlated with long-term recovery of initially injured yet viable tissue components. In conclusion, ARA290 may be a promising therapeutic approach to prevent the conversion of partial- to full-thickness burn injuries. In a clinical setting, the decrease in burn depth and area would likely reduce the necessity for extensive surgical debridement as well as secondary wound closure by means of skin grafting. This use of ARA290 is consistent with its tissue-protective properties previously reported in other models of injury, such as myocardial infarction and hemorrhagic shock. PMID:23401545

  6. LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling.

    PubMed

    Glinka, Andrei; Dolde, Christine; Kirsch, Nadine; Huang, Ya-Lin; Kazanskaya, Olga; Ingelfinger, Dierk; Boutros, Michael; Cruciat, Cristina-Maria; Niehrs, Christof

    2011-10-01

    R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.

  7. Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix.

    PubMed

    Vaday, G G; Franitza, S; Schor, H; Hecht, I; Brill, A; Cahalon, L; Hershkoviz, R; Lider, O

    2001-06-01

    On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)-alpha acts as an adhesion-strengthening and stop signal for T cells migrating toward stromal cell-derived factor-1alpha, while transforming growth factor-beta down-regulates TNF-alpha-induced matrix metalloproteinase-9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF-alpha, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step-by-step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM. PMID:11404372

  8. Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways

    PubMed Central

    2011-01-01

    Background Bone morphogenetic protein (BMP)7 evokes both inductive and axon orienting responses in dorsal interneurons (dI neurons) in the developing spinal cord. These events occur sequentially during the development of spinal neurons but in these and other cell types such inductive and acute chemotactic responses occur concurrently, highlighting the requirement for divergent intracellular signaling. Both type I and type II BMP receptor subtypes have been implicated selectively in orienting responses but it remains unclear how, in a given cell, divergence occurs. We have examined the mechanisms by which disparate BMP7 activities are generated in dorsal spinal neurons. Results We show that widely different threshold concentrations of BMP7 are required to elicit the divergent inductive and axon orienting responses. Type I BMP receptor kinase activity is required for activation of pSmad signaling and induction of dI character by BMP7, a high threshold response. In contrast, neither type I BMP receptor kinase activity nor Smad1/5/8 phosphorylation is involved in the low threshold orienting responses of dI axons to BMP7. Instead, BMP7-evoked axonal repulsion and growth cone collapse are dependent on phosphoinositide-3-kinase (PI3K) activation, plausibly through type II receptor signaling. BMP7 stimulates PI3K-dependent signaling in dI neurons. BMP6, which evokes neural induction but does not have orienting activity, activates Smad signaling but does not stimulate PI3K. Conclusions Divergent signaling through pSmad-dependent and PI3K-dependent (Smad-independent) mechanisms mediates the inductive and orienting responses of dI neurons to BMP7. A model is proposed whereby selective engagement of BMP receptor subunits underlies choice of signaling pathway. PMID:22085733

  9. E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components

    PubMed Central

    Kim, Nam-Gyun; Koh, Eunjin; Chen, Xiao; Gumbiner, Barry M.

    2011-01-01

    Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin–dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell–cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin–bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell–cell adhesion, E-cadherin-mediated cell–cell contact directly regulates the Hippo signaling pathway to control cell proliferation. PMID:21730131

  10. Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

    PubMed Central

    de Jong, Petrus R.; Takahashi, Naoki; Harris, Alexandra R.; Lee, Jihyung; Bertin, Samuel; Jeffries, James; Jung, Michael; Duong, Jen; Triano, Amy I.; Lee, Jongdae; Niv, Yaron; Herdman, David S.; Taniguchi, Koji; Kim, Chang-Whan; Dong, Hui; Eckmann, Lars; Stanford, Stephanie M.; Bottini, Nunzio; Corr, Maripat; Raz, Eyal

    2014-01-01

    The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (ApcMin/+ mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in ApcMin/+ mice, similar to — as well as in conjunction with — a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis. PMID:25083990

  11. Kidney injury molecule-1 (KIM-1) mediates renal epithelial cell repair via ERK MAPK signaling pathway

    PubMed Central

    Zhang, Zhiwei; Cai, Cindy X

    2016-01-01

    The expression of kidney injury molecule-1 (KIM-1), a very promising sensitive and specific urinary biomarker for acute renal injury, is markedly upregulated in injured and regenerating renal proximal tubular epithelial cells following ischemic or toxic insults, suggesting a possible role for this molecule in renal repair process. In the present study we report that expression of KIM-1 facilitates renal tubular epithelial cell repair by promoting cell migration and proliferation. KIM-1 expression also enhances ERK MAPK activation, and the modulatory effect of KIM-1 on cellular repair process is likely mediated via ERK MAPK signaling pathway. PMID:27084535

  12. Multi-organ Site Metastatic Reactivation Mediated by Non-canonical Discoidin Domain Receptor 1 Signaling.

    PubMed

    Gao, Hua; Chakraborty, Goutam; Zhang, Zhanguo; Akalay, Intissar; Gadiya, Mayur; Gao, Yaquan; Sinha, Surajit; Hu, Jian; Jiang, Cizhong; Akram, Muzaffar; Brogi, Edi; Leitinger, Birgit; Giancotti, Filippo G

    2016-06-30

    Genetic screening identifies the atypical tetraspanin TM4SF1 as a strong mediator of metastatic reactivation of breast cancer. Intriguingly, TM4SF1 couples the collagen receptor tyrosine kinase DDR1 to the cortical adaptor syntenin 2 and, hence, to PKCα. The latter kinase phosphorylates and activates JAK2, leading to the activation of STAT3. This non-canonical mechanism of signaling induces the expression of SOX2 and NANOG; sustains the manifestation of cancer stem cell traits; and drives metastatic reactivation in the lung, bone, and brain. Bioinformatic analyses and pathological studies corroborate the clinical relevance of these findings. We conclude that non-canonical DDR1 signaling enables breast cancer cells to exploit the ubiquitous interstitial matrix component collagen I to undergo metastatic reactivation in multiple target organs.

  13. TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity

    PubMed Central

    Ramirez-Ortiz, Zaida G.; Prasad, Amit; Griffith, Jason W.; Pendergraft, William F.; Cowley, Glenn S.; Root, David E.; Tai, Melissa; Luster, Andrew D.; Khoury, Joseph El; Hacohen, Nir; Means, Terry K.

    2015-01-01

    The genes and pathways that fine-tune TLR7-mediated innate inflammatory responses remain to be fully elucidated. Using an unbiased genome-scale shRNA screen, we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4–/– mice were hyporesponsive to TLR7 agonists and failed to produce type I interferon due to impaired phosphorylation of the transcription factor STAT1 by the MAP kinase p38 and decreased recruitment of MyD88 to TLR7. TREML4 deficiency reduced production of inflammatory cytokines and autoantibodies in SLE-prone MRL/lpr mice and inhibited the antiviral immune response to influenza. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity. PMID:25848864

  14. Novel GABAergic circuits mediate the reinforcement-related signals of striatal cholinergic interneurons

    PubMed Central

    English, Daniel F.; Ibanez-Sandoval, Osvaldo; Stark, Eran; Tecuapetla, Fatuel; Buzsaki, Gyorgy; Deisseroth, Karl; Tepper, James M.; Koos, Tibor

    2011-01-01

    Neostriatal cholinergic interneurons are believed to play an important role in reinforcement mediated learning and response selection by signaling the occurrence and motivational value of behaviorally relevant stimuli through precisely timed multiphasic population responses. An important problem is to understand how these signals regulate the functioning of the neostriatum. Here we describe the synaptic organization of a novel circuit that involves direct nicotinic excitation of GABAergic interneurons and enables cholinergic interneurons to exert rapid inhibitory control of the activity of projection neurons. We also demonstrate that the dominant effect of an optogenetically reproduced pause-excitation population response of cholinergic interneurons is powerful and rapid inhibition of the firing of projection neurons that is coincident with synchronous cholinergic activation. These results reveal a previously unknown circuit mechanism that transmits reinforcement-related information of ChAT interneurons in the mouse neostriatal network. PMID:22158514

  15. Stromal cell-mediated glycolytic switch in CLL cells involves Notch-c-Myc signaling.

    PubMed

    Jitschin, Regina; Braun, Martina; Qorraj, Mirjeta; Saul, Domenica; Le Blanc, Katarina; Zenz, Thorsten; Mougiakakos, Dimitrios

    2015-05-28

    It is well established that the stromal niche exerts a protective effect on chronic lymphocytic leukemia (CLL) cells, thereby also affecting their drug sensitivity. One hallmark of malignant cells is metabolic reprogramming, which is mostly represented by a glycolytic shift known as the Warburg effect. Because treatment resistance can be linked to metabolic alterations, we investigated whether bone marrow stromal cells impact the bioenergetics of primary CLL cells. In fact, stromal contact led to an increase of aerobic glycolysis and the cells' overall glycolytic capacity accompanied by an increased glucose uptake, expression of glucose transporter, and glycolytic enzymes. Activation of Notch signaling and of its direct transcriptional target c-Myc contributed to this metabolic switch. Based on these observations, CLL cells' acquired increased glucose dependency as well as Notch-c-Myc signaling could be therapeutically exploited in an effort to overcome stroma-mediated drug resistance.

  16. CD200R/Foxp3-mediated signalling regulates microglial activation

    PubMed Central

    Yi, Min-Hee; Zhang, Enji; Kim, Jwa-Jin; Baek, Hyunjung; Shin, Nara; Kim, Sena; Kim, Sang Ryong; Kim, Hang-Rae; Lee, Sung Joong; Park, Jin Bong; Kim, Yonghyun; Kwon, O-Yu; Lee, Young Ho; Oh, Sang-Ha; Kim, Dong Woon

    2016-01-01

    The heterogeneity of microglial functions have either beneficial or detrimental roles in specific physiological or pathological environments. However, the details of what transcriptional mechanisms induce microglia to take beneficial phenotypes remain unknown. Here, we report that Foxp3 is essential for beneficial outcome of the microglial response and depends upon signalling by the immunoglobulin CD200 through its receptor (CD200R). Foxp3 expression was up-regulated in microglia activated by excitotoxicity-induced hippocampal neuroinflammation. Suppression of CD200R prevented anti-inflammatory phenotype of microglia, but over-expression of Foxp3 enhanced it. Phosphorylation of STAT6, a downstream effector of CD200R, modulated transcription of Foxp3. Finally, CD200R/Foxp3-mediated signalling enhanced hippocampal neuronal viability and conferred a degree of neuroprotection, presumably by counteracting inducible nitric oxide synthase. We conclude that enhancement of Foxp3 through CD200R could be neuroprotective by targeting the microglia. PMID:27731341

  17. 3-iodothyronamine differentially modulates α-2A-adrenergic receptor-mediated signaling.

    PubMed

    Dinter, Juliane; Mühlhaus, Jessica; Jacobi, Simon Friedrich; Wienchol, Carolin Leonie; Cöster, Maxi; Meister, Jaroslawna; Hoefig, Carolin Stephanie; Müller, Anne; Köhrle, Josef; Grüters, Annette; Krude, Heiko; Mittag, Jens; Schöneberg, Torsten; Kleinau, Gunnar; Biebermann, Heike

    2015-06-01

    Most in vivo effects of 3-iodothyronamine (3-T1AM) have been thus far thought to be mediated by binding at the trace amine-associated receptor 1 (TAAR1). Inconsistently, the 3-T1AM-induced hypothermic effect still persists in Taar1 knockout mice, which suggests additional receptor targets. In support of this general assumption, it has previously been reported that 3-T1AM also binds to the α-2A-adrenergic receptor (ADRA2A), which modulates insulin secretion. However, the mechanism of this effect remains unclear. We tested two different scenarios that may explain the effect: the sole action of 3-T1AM at ADRA2A and a combined action of 3-T1AM at ADRA2A and TAAR1, which is also expressed in pancreatic islets. We first investigated a potential general signaling modification using the label-free EPIC technology and then specified changes in signaling by cAMP inhibition and MAPKs (ERK1/2) determination. We found that 3-T1AM induced Gi/o activation at ADRA2A and reduced the norepinephrine (NorEpi)-induced MAPK activation. Interestingly, in ADRA2A/TAAR1 hetero-oligomers, application of NorEpi resulted in uncoupling of the Gi/o signaling pathway, but it did not affect MAPK activation. However, 3-T1AM application in mice over a period of 6 days at a daily dose of 5 mg/kg had no significant effects on glucose homeostasis. In summary, we report an agonistic effect of 3-T1AM on the ADRA2A-mediated Gi/o pathway but an antagonistic effect on MAPK induced by NorEpi. Moreover, in ADRA2A/TAAR1 hetero-oligomers, the capacity of NorEpi to stimulate Gi/o signaling is reduced by co-stimulation with 3-T1AM. The present study therefore points to a complex spectrum of signaling modification mediated by 3-T1AM at different G protein-coupled receptors.

  18. Multiple functionally redundant signals mediate targeting to the apicoplast in the apicomplexan parasite Toxoplasma gondii.

    PubMed

    Harb, Omar S; Chatterjee, Bithi; Fraunholz, Martin J; Crawford, Michael J; Nishi, Manami; Roos, David S

    2004-06-01

    Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle--the apicoplast--acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments. PMID:15189987

  19. Mitochondrial Superoxide Signaling Contributes to Norepinephrine-Mediated T-Lymphocyte Cytokine Profiles

    PubMed Central

    Roessner, Colton T.; Tian, Jun; Zimmerman, Matthew C.

    2016-01-01

    Norepinephrine (NE) produces multifaceted regulatory patterns in T-lymphocytes. Recently, we have shown that NE utilizes redox signaling as evidenced by increased superoxide (O2●-) causally linked to the observed changes in these cells; however, the source of this reactive oxygen species (ROS) remains elusive. Herein, we hypothesized that the source of increased O2●- in NE-stimulated T-lymphocytes is due to disruption of mitochondrial bioenergetics. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T-lymphocytes stimulated with NE and assessed O2●- levels, mitochondrial metabolism, cellular proliferation, and cytokine profiles. We demonstrate that the increase in O2●- levels in response to NE is time-dependent and occurs at later points of T-lymphocyte activation. Moreover, the source of O2●- was indeed the mitochondria as evidenced by enhanced MitoSOX Red oxidation as well as abrogation of this signal by the addition of the mitochondrial-targeted O2●--scavenging antioxidant MitoTempol. NE-stimulated T-lymphocytes also demonstrated decreased mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2●-. The effects of NE in regards to redox signaling appear to be adrenergic receptor-dependent as specific receptor antagonists could reverse the increase in O2●-; however, differential receptors regulating these processes were observed in CD4+ versus CD8+ T-lymphocytes. Finally, mitochondrial O2●- was shown to be mechanistic to the NE-mediated T-lymphocyte phenotype as supplementation of MitoTempol could reverse specific changes in cytokine expression observed with NE treatment. Overall, these studies indicate that mitochondrial metabolism and O2●--mediated redox signaling play a regulatory role in the T-lymphocyte response to NE. PMID:27727316

  20. Phosphatidic acid mediates activation of mTORC1 through the ERK signaling pathway

    PubMed Central

    Winter, Jeremiah N.; Fox, Todd E.; Kester, Mark; Jefferson, Leonard S.

    2010-01-01

    The mammalian target of rapamycin (mTOR) assembles into two distinct multiprotein complexes known as mTORC1 and mTORC2. Of the two complexes, mTORC1 acts to integrate a variety of positive and negative signals to downstream targets that regulate cell growth. The lipid second messenger, phosphatidic acid (PA), represents one positive input to mTORC1, and it is thought to act by binding directly to mTOR, thereby enhancing the protein kinase activity of mTORC1. Support for this model includes findings that PA binds directly to mTOR and addition of PA to the medium of cells in culture results in activation of mTORC1. In contrast, the results of the present study do not support a model in which PA activates mTORC1 through direct interaction with the protein kinase but, instead, show that the lipid promotes mTORC1 signaling through activation of the ERK pathway. Moreover, rather than acting directly on mTORC1, the results suggest that exogenous PA must be metabolized to lysophosphatidic acid (LPA), which subsequently activates the LPA receptor endothelial differentiation gene (EDG-2). Finally, in contrast to previous studies, the results of the present study demonstrate that leucine does not act through phospholipase D and PA to activate mTORC1 and, instead, show that the two mediators act through parallel upstream signaling pathways to activate mTORC1. Overall, the results demonstrate that leucine and PA signal through parallel pathways to activate mTORC1 and that PA mediates its effect through the ERK pathway, rather than through direct binding to mTOR. PMID:20427710

  1. Phospholipase D signaling mediates reactive oxygen species-induced lung endothelial barrier dysfunction.

    PubMed

    Usatyuk, Peter V; Kotha, Sainath R; Parinandi, Narasimham L; Natarajan, Viswanathan

    2013-01-01

    Reactive oxygen species (ROS) have emerged as critical players in the pathophysiology of pulmonary disorders and diseases. Earlier, we have demonstrated that ROS stimulate lung endothelial cell (EC) phospholipase D (PLD) that generates phosphatidic acid (PA), a second messenger involved in signal transduction. In the current study, we investigated the role of PLD signaling in the ROS-induced lung vascular EC barrier dysfunction. Our results demonstrated that hydrogen peroxide (H2O2), a typical physiological ROS, induced PLD activation and altered the barrier function in bovine pulmonary artery ECs (BPAECs). 1-Butanol, the quencher of PLD, generated PA leading to the formation of physiologically inactive phosphatidyl butanol but not its biologically inactive analog, 2-butanol, blocked the H2O2-mediated barrier dysfunction. Furthermore, cell permeable C2 ceramide, an inhibitor of PLD but not the C2 dihydroceramide, attenuated the H2O2-induced PLD activation and enhancement of paracellular permeability of Evans blue conjugated albumin across the BPAEC monolayers. In addition, transfection of BPAECs with adenoviral constructs of hPLD1 and mPLD2 mutants attenuated the H2O2-induced barrier dysfunction, cytoskeletal reorganization and distribution of focal adhesion proteins. For the first time, this study demonstrated that the PLD-generated intracellular bioactive lipid signal mediator, PA, played a critical role in the ROS-induced barrier dysfunction in lung vascular ECs. This study also underscores the importance of PLD signaling in vascular leak and associated tissue injury in the etiology of lung diseases among critically ill patients encountering oxygen toxicity and excess ROS production during ventilator-assisted breathing.

  2. CD5-mediated inhibition of TCR signaling proceeds normally in the absence of SHP-1

    PubMed Central

    DONG, BAOXIA; SOMANI, ALLY-KHAN; LOVE, PAUL E.; ZHENG, XUAN; CHEN, XIEQUN; ZHANG, JINYI

    2016-01-01

    The CD5 transmembrane glycoprotein functions as a co-receptor in the signaling pathway linking T-cell antigen receptor (TCR) engagement to activation and differentiation. Although CD5 effects on TCR signaling have been shown to be primarily inhibitory, the underlying mechanisms remain unclear. In view of recent data revealing the ability of CD5 to associate with the SHP-1 tyrosine phosphatase, a protein that also downregulates TCR signaling, we examined the role of SHP-1 in modulating CD5 function using thymocytes from SHP-1-deficient viable motheaten (mev) mice. The results revealed the association of SHP-1 with CD5 to be markedly increased following TCR stimulation and indicated that this interaction was enhanced by and was dependent on CD5 tyrosine phosphorylation. However, there was no difference of the tyrosine phosphorylation status of CD5 between resting and TCR-stimulated cells in SHP-1-deficient compared to wild-type thymocytes. Lack of SHP-1 activity did not affect the levels of CD5 surface expression, CD5 co-immunoprecipitable tyrosine phosphatase activity and intracellular calcium increase following co-crosslinking of the TCR and CD5. Similarly, an analysis of T-cell thymocyte populations in mev mice expressing an H-Y transgene as well as a construct mediating T-cell restricted CD5 overexpression, revealed that the reduction in the positive selection conferred by CD5 overexpression was unaffected by SHP-1 deficiency. CD5 is not a SHP-1 substrate and SHP-1 is not required for and possibly not involved in the CD5-mediated modulation of TCR signaling. PMID:27221212

  3. Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells

    PubMed Central

    Pérez-Pérez, Antonio; Sánchez-Jiménez, Flora; Vilariño-García, Teresa; de la Cruz, Luis; Virizuela, Juan A.; Sánchez-Margalet, Víctor

    2016-01-01

    Obesity is a well-known risk factor for breast cancer development in postmenopausal women. High insulin and leptin levels seem to have a role modulating the growth of these tumours. Sam68 is an RNA-binding protein with signalling functions that has been found to be overexpressed in breast cancer. Moreover, Sam68 may be recruited to insulin and leptin signalling pathways, mediating its effects on survival, growth and proliferation in different cellular types. We aimed to study the expression of Sam68 and its phosphorylation level upon insulin and leptin stimulation, and the role of Sam68 in the proliferative effect and signalling pathways that are activated by insulin or leptin in human breast adenocarcinoma cells. In the human breast adenocarcinoma cell lines MCF7, MDA-MB-231 and BT-474, Sam68 protein quantity and gene expression were increased upon leptin or insulin stimulation, as it was checked by qPCR and immunoblot. Moreover, both insulin and leptin stimulation promoted an increase in Sam68 tyrosine phosphorylation and negatively regulated its RNA binding capacity. siRNA was used to downregulate Sam68 expression, which resulted in lower proliferative effects of both insulin and leptin, as well as a lower activation of MAPK and PI3K pathways promoted by both hormones. These effects may be partly explained by the decrease in IRS-1 expression by down-regulation of Sam68. These results suggest the participation of Sam68 in both leptin and insulin receptor signaling in human breast cancer cells, mediating the trophic effects of these hormones in proliferation and cellular growth. PMID:27415018

  4. CYP1B1: a unique gene with unique characteristics.

    PubMed

    Faiq, Muneeb A; Dada, Rima; Sharma, Reetika; Saluja, Daman; Dada, Tanuj

    2014-01-01

    CYP1B1, a recently described dioxin inducible oxidoreductase, is a member of the cytochrome P450 superfamily involved in the metabolism of estradiol, retinol, benzo[a]pyrene, tamoxifen, melatonin, sterols etc. It plays important roles in numerous physiological processes and is expressed at mRNA level in many tissues and anatomical compartments. CYP1B1 has been implicated in scores of disorders. Analyses of the recent studies suggest that CYP1B1 can serve as a universal/ideal cancer marker and a candidate gene for predictive diagnosis. There is plethora of literature available about certain aspects of CYP1B1 that have not been interpreted, discussed and philosophized upon. The present analysis examines CYP1B1 as a peculiar gene with certain distinctive characteristics like the uniqueness in its chromosomal location, gene structure and organization, involvement in developmentally important disorders, tissue specific, not only expression, but splicing, potential as a universal cancer marker due to its involvement in key aspects of cellular metabolism, use in diagnosis and predictive diagnosis of various diseases and the importance and function of CYP1B1 mRNA in addition to the regular translation. Also CYP1B1 is very difficult to express in heterologous expression systems, thereby, halting its functional studies. Here we review and analyze these exceptional and startling characteristics of CYP1B1 with inputs from our own experiences in order to get a better insight into its molecular biology in health and disease. This may help to further understand the etiopathomechanistic aspects of CYP1B1 mediated diseases paving way for better research strategies and improved clinical management. PMID:25658124

  5. Essential role of endocytosis for interleukin-4-receptor-mediated JAK/STAT signalling.

    PubMed

    Kurgonaite, Kristina; Gandhi, Hetvi; Kurth, Thomas; Pautot, Sophie; Schwille, Petra; Weidemann, Thomas; Bökel, Christian

    2015-10-15

    Many important signalling cascades operate through specialized signalling endosomes, but a corresponding mechanism has as yet not been described for hematopoietic cytokine receptors. Based on live-cell affinity measurements, we recently proposed that ligand-induced interleukin-4 receptor (IL-4R) complex formation and thus JAK/STAT pathway activation requires a local subcellular increase in receptor density. Here, we show that this concentration step is provided by the internalization of IL-4R subunits through a constitutive, Rac1-, Pak- and actin-mediated endocytosis route that causes IL-4R subunits to become enriched by about two orders of magnitude within a population of cortical endosomes. Consistently, ligand-induced receptor dimers are preferentially detected within these endosomes. IL-4 signalling can be blocked by pharmacological inhibitors targeting the actin polymerization machinery driving receptor internalization, placing endocytosis unambigously upstream of receptor activation. Taken together, these observations demonstrate a role for endocytosis that is mechanistically distinct from the scaffolding function of signalling endosomes in other pathways.

  6. Requirement of Smad4-mediated signaling in odontoblast differentiation and dentin matrix formation

    PubMed Central

    Yun, Chi-Young; Choi, Hwajung; You, Young-Jae; Yang, Jin-Young

    2016-01-01

    Dentin is the major part of tooth and formed by odontoblasts. Under the influence of the inner enamel epithelium, odontoblasts differentiate from ectomesenchymal cells of the dental papilla and secrete pre-dentin which then undergo mineralization into dentin. Transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) signaling is essential for dentinogenesis; however, the precise molecular mechanisms remain unclear. To understand the role of TGF-β/BMP signaling in odontoblast differentiation and dentin formation, we generated mice with conditional ablation of Smad4, a key intracellular mediator of TGF-β/BMP signaling, using Osr2 or OC-Cre mice. Here we found the molars of Osr2CreSmad4 mutant mice exhibited impaired odontoblast differentiation, and normal dentin was replaced by ectopic bone-like structure. In Osr2CreSmad4 mutant mice, cell polarity of odontoblast was lost, and the thickness of crown dentin was decreased in later stage compared to wild type. Moreover, the root dentin was also impaired and showed ectopic bone-like structure similar to Osr2CreSmad4 mutant mice. Taken together, our results suggest that Smad4-dependent TGF-β/BMP signaling plays a critical role in odontoblast differentiation and dentin formation during tooth development. PMID:27722013

  7. Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress.

    PubMed

    Evanson, Nathan K; Herman, James P

    2015-10-15

    Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus-pituitary-adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

  8. Yeast as a model system to study glucose-mediated signalling and response.

    PubMed

    Sanz, Pascual

    2007-01-01

    Glucose is the principal carbon and energy source for a wide variety of cells, ranging from unicellular microorganisms to higher eukaryotic cells. It is taken up by these cells and metabolized to obtain the energy necessary for cell viability. In addition, the presence of this sugar is able to adjust cellular metabolism, regulate gene expression and even influence cell growth. For this reason, glucose is considered as a "hormone". Specifically, it can trigger different signalling pathways that allow cells to adjust their gene expression programmes in response to glucose availability. Elucidating the molecular mechanisms of glucose response in eukaryotes has been greatly aided by studies conducted in the yeast Saccharomyces cerevisiae. This yeast shares with complex multicellular eukaryotes many of the signal transduction components that detect glucose, transmit the corresponding signals to the interior of the cell and make the needed adjustments to cellular metabolism and gene expression. In this manuscript, I will review the current knowledge of some aspects of glucose-mediated signalling in yeast and discuss how these results have contributed to the understanding of similar processes in mammalian cells.

  9. Nonclassical estrogen receptor alpha signaling mediates negative feedback in the female mouse reproductive axis.

    PubMed

    Glidewell-Kenney, C; Hurley, L A; Pfaff, L; Weiss, J; Levine, J E; Jameson, J L

    2007-05-01

    Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) alpha but not ERbeta knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERalpha. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERalpha signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERalpha (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERalpha-null (-/-) background, we examine the ability of ERE-independent ERalpha signaling pathways to convey estrogen feedback regulation of the female hypothalamic-pituitary axis in vivo. ERalpha-/AA exhibited 69.9% lower serum LH levels compared with ERalpha-/- mice. Additionally, like wild type, ERalpha-/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERalpha-/AA mice. However, unlike wild type, both ERalpha-/AA and ERalpha-/- mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERalpha signaling is sufficient to convey a major portion of estrogen's negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERalpha signaling.

  10. mTORC1 is a critical mediator of oncogenic Semaphorin3A signaling.

    PubMed

    Yamada, Daisuke; Kawahara, Kohichi; Maeda, Takehiko

    2016-08-01

    Aberration of signaling pathways by genetic mutations or alterations in the surrounding tissue environments can result in tumor development or metastasis. However, signaling molecules responsible for these processes have not been completely elucidated. Here, we used mouse Lewis lung carcinoma cells (LLC) to explore the mechanism by which the oncogenic activity of Semaphorin3A (Sema3A) signaling is regulated. Sema3A knockdown by shRNA did not affect apoptosis, but decreased cell proliferation in LLCs; both the mammalian target of rapamycin complex 1 (mTORC1) level and glycolytic activity were also decreased. In addition, Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation by oligomycin, but conferred resistance to decreased cell viability induced by glucose starvation. Furthermore, recombinant SEMA3A rescued the attenuation of cell proliferation and glycolytic activity in LLCs after Sema3A knockdown, whereas mTORC1 inhibition by rapamycin completely counteracted this effect. These results demonstrate that Sema3A signaling exerts its oncogenic effect by promoting an mTORC1-mediated metabolic shift from oxidative phosphorylation to aerobic glycolysis. PMID:27246732

  11. The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

    PubMed Central

    Baker, Sandra M.; Loren, Cassandra P.; Haley, Kristina M.; Itakura, Asako; Pang, Jiaqing; Greenberg, Daniel L.; David, Larry L.; Manser, Ed; Chernoff, Jonathan; McCarty, Owen J. T.

    2013-01-01

    Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets. PMID:23784547

  12. Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis.

    PubMed

    Li, Jianzhong; Ren, Jiafa; Liu, Xin; Jiang, Lei; He, Weichun; Yuan, Weiping; Yang, Junwei; Dai, Chunsun

    2015-09-01

    The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases.

  13. MCPIP1 endoribonuclease activity negatively regulates interleukin-17-mediated signaling and inflammation

    PubMed Central

    Garg, Abhishek V.; Amatya, Nilesh; Chen, Kong; Cruz, J. Agustin; Grover, Prerna; Whibley, Natasha; Conti, Heather R.; Mir, Gerard Hernandez; Sirakova, Tatiana; Childs, Erin C.; Smithgall, Thomas E.; Biswas, Partha S.; Kolls, Jay K.; McGeachy, Mandy J.; Kolattukudy, Pappachan E.; Gaffen, Sarah L.

    2015-01-01

    SUMMARY Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1’s endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra−/− background. Conversely, IL-17-dependent pathology in Zc3h12a+/− mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly, but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3’ UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling. PMID:26320658

  14. Shared functional defect in IP3R-mediated calcium signaling in diverse monogenic autism syndromes

    PubMed Central

    Schmunk, G; Boubion, B J; Smith, I F; Parker, I; Gargus, J J

    2015-01-01

    Autism spectrum disorder (ASD) affects 2% of children, and is characterized by impaired social and communication skills together with repetitive, stereotypic behavior. The pathophysiology of ASD is complex due to genetic and environmental heterogeneity, complicating the development of therapies and making diagnosis challenging. Growing genetic evidence supports a role of disrupted Ca2+ signaling in ASD. Here, we report that patient-derived fibroblasts from three monogenic models of ASD—fragile X and tuberous sclerosis TSC1 and TSC2 syndromes—display depressed Ca2+ release through inositol trisphosphate receptors (IP3Rs). This was apparent in Ca2+ signals evoked by G protein-coupled receptors and by photoreleased IP3 at the levels of both global and local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given the ubiquitous involvement of IP3R-mediated Ca2+ signaling in neuronal excitability, synaptic plasticity, gene expression and neurodevelopment, we propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect. These findings highlight potential pharmaceutical targets, and identify Ca2+ screening in skin fibroblasts as a promising technique for early detection of individuals susceptible to ASD. PMID:26393489

  15. Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis

    PubMed Central

    Li, Jianzhong; Ren, Jiafa; Liu, Xin; Jiang, Lei; He, Weichun; Yuan, Weiping; Yang, Junwei; Dai, Chunsun

    2015-01-01

    The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases. PMID:25970154

  16. Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.

    PubMed

    Ising, Christina; Koehler, Sybille; Brähler, Sebastian; Merkwirth, Carsten; Höhne, Martin; Baris, Olivier R; Hagmann, Henning; Kann, Martin; Fabretti, Francesca; Dafinger, Claudia; Bloch, Wilhelm; Schermer, Bernhard; Linkermann, Andreas; Brüning, Jens C; Kurschat, Christine E; Müller, Roman-Ulrich; Wiesner, Rudolf J; Langer, Thomas; Benzing, Thomas; Brinkkoetter, Paul Thomas

    2015-02-02

    Mitochondrial dysfunction and alterations in energy metabolism have been implicated in a variety of human diseases. Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Here, we provide a link between PHB2 deficiency and hyperactive insulin/IGF-1 signaling. Deletion of PHB2 in podocytes of mice, terminally differentiated cells at the kidney filtration barrier, caused progressive proteinuria, kidney failure, and death of the animals and resulted in hyperphosphorylation of S6 ribosomal protein (S6RP), a known mediator of the mTOR signaling pathway. Inhibition of the insulin/IGF-1 signaling system through genetic deletion of the insulin receptor alone or in combination with the IGF-1 receptor or treatment with rapamycin prevented hyperphosphorylation of S6RP without affecting the mitochondrial structural defect, alleviated renal disease, and delayed the onset of kidney failure in PHB2-deficient animals. Evidently, perturbation of insulin/IGF-1 receptor signaling contributes to tissue damage in mitochondrial disease, which may allow therapeutic intervention against a wide spectrum of diseases.

  17. Restoration of β -Adrenergic Signaling in Failing Cardiac Ventricular Myocytes via Adenoviral-Mediated Gene Transfer

    NASA Astrophysics Data System (ADS)

    Akhter, Shahab A.; Skaer, Christine A.; Kypson, Alan P.; McDonald, Patricia H.; Peppel, Karsten C.; Glower, Donald D.; Lefkowitz, Robert J.; Koch, Walter J.

    1997-10-01

    Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β -adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β -adrenergic signaling defects including down-regulation of myocardial β -adrenergic receptors (β -ARs), functional β -AR uncoupling, and an upregulation of the β -AR kinase (β ARK1). Adenoviral-mediated gene transfer of the human β 2-AR or an inhibitor of β ARK1 to these failing myocytes led to the restoration of β -AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of β ARK1 activity in the heart.

  18. Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

    PubMed

    Michel, Laurent; Bachelard, Aude; Reimmann, Cornelia

    2007-05-01

    In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

  19. Tight regulation of diacylglycerol-mediated signaling is critical for proper invariant NKT cell development

    PubMed Central

    Shen, Shudan; Wu, Jinhong; Srivatsan, Sruti; Gorentla, Balachandra; Shin, Jinwook; Xu, Li; Zhong, Xiao-Ping

    2011-01-01

    Type I natural killer T (NKT) cells, or iNKT cells, express a semi-invariant T cell receptor characterized by its unique V α 14-Jα 18 usage (iV α 14TCR). Upon interaction with glycolipid/CD1d complexes, the iV α 14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the PKCθ-IKKα/β-NFκB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases (DGKs) play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. Here we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NFκB and Ras-Erk1/2 activation. Moreover, constitutive IKKβ and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development. PMID:21775687

  20. p53 mediates impaired insulin signaling in 3T3-L1 adipocytes during hyperinsulinemia.

    PubMed

    Posa, Jyothi Kumari; Selvaraj, Sudhagar; Sangeetha, K N; Baskaran, Sarath Kumar; Lakshmi, B S

    2014-07-01

    Hyperinsulinemia is being implicated in the development of insulin resistance but remains poorly understood. The present study focuses on p53-mediated impaired insulin signaling by hyperinsulinemia in 3T3-L1 adipocytes. Hyperinsulinemia impairs insulin-stimulated glucose uptake and its cellular signaling in a dose- and time-dependent manner. An increased level of reactive oxygen species (ROS) and stress response signals were observed, and quenching of the ROS by an antioxidant N-acetylcysteine (NAC) did not revert impaired insulin sensitivity. The tumor suppressor p53 has emerged as a crucial factor in the metabolic adaptation of cancer cells under nutritional starvation and is being studied in the development of insulin resistance in adipocytes at physiological level. Interestingly, we observed hyperinsulinemia-enhanced p53 level in a time-dependent manner without exhibiting cytotoxicity. Transient knockdown of p53 partially improved insulin sensitivity revealing a novel link between p53 and insulin signaling in adipocytes. The findings suggest that hyperinsulinemia-induced p53 impairs insulin sensitivity in 3T3-L1 adipocytes.

  1. The transrepression arm of glucocorticoid receptor signaling is protective in mutant huntingtin-mediated neurodegeneration.

    PubMed

    Varadarajan, S; Breda, C; Smalley, J L; Butterworth, M; Farrow, S N; Giorgini, F; Cohen, G M

    2015-08-01

    The unfolded protein response (UPR) occurs following the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and orchestrates an intricate balance between its prosurvival and apoptotic arms to restore cellular homeostasis and integrity. However, in certain neurodegenerative diseases, the apoptotic arm of the UPR is enhanced, resulting in excessive neuronal cell death and disease progression, both of which can be overcome by modulating the UPR. Here, we describe a novel crosstalk between glucocorticoid receptor signaling and the apoptotic arm of the UPR, thus highlighting the potential of glucocorticoid therapy in treating neurodegenerative diseases. Several glucocorticoids, but not mineralocorticoids, selectively antagonize ER stress-induced apoptosis in a manner that is downstream of and/or independent of the conventional UPR pathways. Using GRT10, a novel selective pharmacological modulator of glucocorticoid signaling, we describe the importance of the transrepression arm of the glucocorticoid signaling pathway in protection against ER stress-induced apoptosis. Furthermore, we also observe the protective effects of glucocorticoids in vivo in a Drosophila model of Huntington's disease (HD), wherein treatment with different glucocorticoids diminished rhabdomere loss and conferred neuroprotection. Finally, we find that growth differentiation factor 15 has an important role downstream of glucocorticoid signaling in antagonizing ER stress-induced apoptosis in cells, as well as in preventing HD-mediated neurodegeneration in flies. Thus, our studies demonstrate that this novel crosstalk has the potential to be effectively exploited in alleviating several neurodegenerative disorders.

  2. BMP-FGF Signaling Axis Mediates Wnt-Induced Epidermal Stratification in Developing Mammalian Skin

    PubMed Central

    Zhu, Xiao-Jing; Liu, YuDong; Dai, Zhong-Min; Zhang, Xiaoyun; Yang, XueQin; Li, Yan; Qiu, Mengsheng; Fu, Jiang; Hsu, Wei; Chen, YiPing; Zhang, Zunyi

    2014-01-01

    Epidermal stratification of the mammalian skin requires proliferative basal progenitors to generate intermediate cells that separate from the basal layer and are replaced by post-mitotic cells. Although Wnt signaling has been implicated in this developmental process, the mechanism underlying Wnt-mediated regulation of basal progenitors remains elusive. Here we show that Wnt secreted from proliferative basal cells is not required for their differentiation. However, epidermal production of Wnts is essential for the formation of the spinous layer through modulation of a BMP-FGF signaling cascade in the dermis. The spinous layer defects caused by disruption of Wnt secretion can be restored by transgenically expressed Bmp4. Non-cell autonomous BMP4 promotes activation of FGF7 and FGF10 signaling, leading to an increase in proliferative basal cell population. Our findings identify an essential BMP-FGF signaling axis in the dermis that responds to the epidermal Wnts and feedbacks to regulate basal progenitors during epidermal stratification. PMID:25329657

  3. Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway

    SciTech Connect

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Won, Seong Su; Kang, Wonku; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-05-15

    Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1 expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells. - Highlights: • Leptin increased 4-hydroxyoestradiol in MCF-7 breast cancer cells. • Leptin activated ERK and Akt kinases related to ERα phosphorylation. • Leptin induces phosphorylation of ERα at serine residues 118 and 167. • Leptin induces ERE-luciferase activity.

  4. Is insulin signaling molecules misguided in diabetes for ubiquitin-proteasome mediated degradation?

    PubMed

    Balasubramanyam, Muthuswamy; Sampathkumar, Rangasamy; Mohan, Viswanathan

    2005-07-01

    Recent mining of the human and mouse genomes, use of yeast genetics, and detailed analyses of several biochemical pathways, have resulted in the identification of many new roles for ubiquitin-proteasome mediated degradation of proteins. In the context of last year's award of Noble Prize (Chemistry) work, the ubiquitin and ubiquitin-like modifications are increasingly recognized as key regulatory events in health and disease. Although the ATP-dependent ubiquitin-proteasome system has evolved as premier cellular proteolytic machinery, dysregulation of this system by several different mechanisms leads to inappropriate degradation of specific proteins and pathological consequences. While aberrations in the ubiquitin-proteasome pathway have been implicated in certain malignancies and neurodegenerative disorders, recent studies indicate a role for this system in the pathogenesis of diabetes and its complications. Inappropriate degradation of insulin signaling molecules such as insulin receptor substrates (IRS-1 and IRS-2) has been demonstrated in experimental diabetes, mediated in part through the up-regulation of suppressors of cytokine signaling (SOCS). It appears that altered ubiquitin-proteasome system might be one of the molecular mechanisms of insulin resistance in many pathological situations. Drugs that modulate the SOCS action and/or proteasomal degradation of proteins could become novel agents for the treatment of insulin resistance and Type 2 diabetes.

  5. β-Arrestin1 enhances hepatocellular carcinogenesis through inflammation-mediated Akt signalling.

    PubMed

    Yang, Yidong; Guo, Yunwei; Tan, Siwei; Ke, Bilun; Tao, Jin; Liu, Huiling; Jiang, Jie; Chen, Jianning; Chen, Guihua; Wu, Bin

    2015-01-01

    G-protein-coupled receptors (GPCR) constitute the largest known superfamily for signal transduction and transmission, and they control a variety of physiological and pathological processes. GPCR adaptor β-arrestins (ARRBs) play a role in cancerous proliferation. However, the effect of ARRBs in inflammation-mediated hepatocellular carcinogenesis is unknown. Here we show that ARRB1, but not ARRB2, is upregulated in inflammation-associated hepatocellular carcinoma (HCC) and paracancerous tissues in humans. A genotoxic carcinogen, diethylnitrosamine (DEN), significantly induces hepatic inflammation, TNF-α production and ARRB1 expression. Although ARRB1 deficiency does not affect hepatic inflammation and TNF-α production, it markedly represses hepatocellular carcinogenesis by suppressing malignant proliferation in DEN-treated mice. Furthermore, TNF-α directly induces hepatic ARRB1 expression and enhances ARRB1 interaction with Akt by binding to boost Akt phosphorylation, resulting in malignant proliferation of liver cells. Our data suggest that ARRB1 enhances hepatocellular carcinogenesis by inflammation-mediated Akt signalling and that ARRB1 may be a potential therapeutic target for HCC.

  6. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

    NASA Technical Reports Server (NTRS)

    Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

  7. SET9-Mediated Regulation of TGF-β Signaling Links Protein Methylation to Pulmonary Fibrosis.

    PubMed

    Elkouris, Maximilianos; Kontaki, Haroula; Stavropoulos, Athanasios; Antonoglou, Anastasia; Nikolaou, Kostas C; Samiotaki, Martina; Szantai, Eszter; Saviolaki, Dimitra; Brown, Peter J; Sideras, Paschalis; Panayotou, George; Talianidis, Iannis

    2016-06-21

    TGF-β signaling regulates a variety of cellular processes, including proliferation, apoptosis, differentiation, immune responses, and fibrogenesis. Here, we describe a lysine methylation-mediated mechanism that controls the pro-fibrogenic activity of TGF-β. We find that the methyltransferase Set9 potentiates TGF-β signaling by targeting Smad7, an inhibitory downstream effector. Smad7 methylation promotes interaction with the E3 ligase Arkadia and, thus, ubiquitination-dependent degradation. Depletion or pharmacological inhibition of Set9 results in elevated Smad7 protein levels and inhibits TGF-β-dependent expression of genes encoding extracellular matrix components. The inhibitory effect of Set9 on TGF-β-mediated extracellular matrix production is further demonstrated in mouse models of pulmonary fibrosis. Lung fibrosis induced by bleomycin or Ad-TGF-β treatment was highly compromised in Set9-deficient mice. These results uncover a complex regulatory interplay among multiple Smad7 modifications and highlight the possibility that protein methyltransferases may represent promising therapeutic targets for treating lung fibrosis. PMID:27292644

  8. FLIP switches Fas-mediated glucose signaling in human pancreatic cells from apoptosis to cell replication

    NASA Astrophysics Data System (ADS)

    Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

    2002-06-01

    Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of cell turnover. In human islets, elevated glucose concentrations impair cell proliferation and induce cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells; FLIP was no longer detectable in such TUNEL-positive cells. Up-regulation of FLIP, by incubation with transforming growth factor or by transfection with an expression vector coding for FLIP, protected cells from glucose-induced apoptosis, restored cell proliferation, and improved cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

  9. 18 CFR 1b.1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Section 1b.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.1 Definitions. For purposes of this part— (a... pipelines, electric utilities and hydroelectric projects....

  10. 18 CFR 1b.1 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Section 1b.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.1 Definitions. For purposes of this part— (a... pipelines, electric utilities and hydroelectric projects....

  11. 18 CFR 1b.1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Section 1b.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.1 Definitions. For purposes of this part— (a... pipelines, electric utilities and hydroelectric projects....

  12. 18 CFR 1b.1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Section 1b.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.1 Definitions. For purposes of this part— (a... pipelines, electric utilities and hydroelectric projects....

  13. 18 CFR 1b.1 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Section 1b.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.1 Definitions. For purposes of this part— (a... pipelines, electric utilities and hydroelectric projects....

  14. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

    PubMed Central

    2011-01-01

    Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when

  15. Niacin activates the G protein estrogen receptor (GPER)-mediated signalling.

    PubMed

    Santolla, Maria Francesca; De Francesco, Ernestina Marianna; Lappano, Rosamaria; Rosano, Camillo; Abonante, Sergio; Maggiolini, Marcello

    2014-07-01

    Nicotinic acid, also known as niacin, is the water soluble vitamin B3 used for decades for the treatment of dyslipidemic diseases. Its action is mainly mediated by the G protein-coupled receptor (GPR) 109A; however, certain regulatory effects on lipid levels occur in a GPR109A-independent manner. The amide form of nicotinic acid, named nicotinamide, acts as a vitamin although neither activates the GPR109A nor exhibits the pharmacological properties of nicotinic acid. In the present study, we demonstrate for the first time that nicotinic acid and nicotinamide bind to and activate the GPER-mediated signalling in breast cancer cells and cancer-associated fibroblasts (CAFs). In particular, we show that both molecules are able to promote the up-regulation of well established GPER target genes through the EGFR/ERK transduction pathway. As a biological counterpart, nicotinic acid and nicotinamide induce proliferative and migratory effects in breast cancer cells and CAFs in a GPER-dependent fashion. Moreover, nicotinic acid prevents the up-regulation of ICAM-1 triggered by the pro-inflammatory cytokine TNF-α and stimulates the formation of endothelial tubes through GPER in HUVECs. Together, our findings concerning the agonist activity for GPER displayed by both nicotinic acid and nicotinamide broaden the mechanisms involved in the biological action of these molecules and further support the potential of a ligand to induce different responses mediated in a promiscuous manner by distinct GPCRs.

  16. Structural modeling and analysis of dengue-mediated inhibition of interferon signaling pathway.

    PubMed

    Aslam, B; Ahmad, J; Ali, A; Paracha, R Z; Tareen, S H K; Khusro, S; Ahmad, T; Muhammad, S A; Niazi, U; Azevedo, V

    2015-01-01

    Dengue virus (DENV) belongs to the family Flaviviridae and can cause major health problems worldwide, including dengue fever and dengue shock syndrome. DENV replicon in human cells inhibits interferon α and β with the help of its non-structural proteins. Non-structural protein 5 (NS5) of DENV is responsible for the proteasome-mediated degradation of signal transducer and activator of transcription (STAT) 2 protein, which has been implicated in the development of resistance against interferon-mediated antiviral effect. This degradation of STAT2 primarily occurs with the help of E3 ubiquitin ligases. Seven in absentia homologue (SIAH) 2 is a host protein that can mediate the ubiquitination of proteins and is known for its interaction with NS5. In this study, comprehensive computational analysis was performed to characterize the protein-protein interactions between NS5, SIAH2, and STAT2 to gain insight into the residues and sites of interaction between these proteins. The objective of the study was to structurally characterize the NS5-STAT2, SIAH2-STAT2, and NS5-SIAH2 interactions along with the determination of the possible reaction pattern for the degradation of STAT2. Docking and physicochemical studies indicated that DENV NS5 may first interact with the host SIAH2, which can then proceed towards binding with STAT2 from the side of SIAH2. These implications are reported for the first time and require validation by wet-lab studies.

  17. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    SciTech Connect

    Zheng, Long-Yi; Zhou, Dong-Xun; Lu, Jin; Zhang, Wen-Jun; Zou, Da-Jin

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer PTP1B protein showed decreased expression in 67.79% of the HCC patients. Black-Right-Pointing-Pointer Low PTP1B expression predicts poor prognosis of HCC. Black-Right-Pointing-Pointer Low PTP1B expression is correlated with expansion of OV6{sup +} tumor-initiating cells. Black-Right-Pointing-Pointer Down-regulation of PTP1B is associated with activation of Wnt/{beta}-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6{sup +} tumor-initiating cells (T-ICs) and high frequency of nuclear {beta}-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/{beta}-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  18. Active ERK2 is sufficient to mediate growth arrest and differentiation signaling

    PubMed Central

    Wu, Pui-Kei; Hong, Seung-Keun; Yoon, Seung-Hee; Park, Jong-In

    2015-01-01

    Although extracellular signal-regulated kinase (ERK) ½ has been shown for its necessity for a variety of the Raf/MEK/ERK pathway signaling, its sufficiency in mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously reported ERK2 mutants that exhibit enhanced activity of autophosphorylation of TEY sites in the activation loop for their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. Here, we demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp replacements that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation whereas ERK2-I84A and ERK2-R65S/D319N are not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D could only mildly increase ERK kinase activity in cells, as determined by the ERK substrates, p90RSK and ELK1. Nevertheless, ERK2-L73P/S151D expression effectively induced downregulation of androgen receptor, Rb and E2F1, and upregulation of p16INK4A and p21CIP1, which were accompanied by cell cycle arrest and morphological differentiation, in LNCaP cells and neurite-like processing in PC12 cells. These effects and TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introducing the active site-disabling Lys52Arg mutation, confirming its sufficiency in this signaling. Moreover, introduction of the mutations (producing Asp316/319Ala or Asp319Asn) that impair the common docking site/D-domain-based physical interaction of ERK did not significantly affect the ERK2-L73P/S151D signaling, suggesting that ERK2 can mediate growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Our study presents a convincing example of ERK sufficiency for Raf/MEK/ERK signaling. PMID:25639353

  19. CXCR2 Signaling Protects Oligodendrocyte Progenitor Cells from IFN-γ/CXCL10-Mediated Apoptosis

    PubMed Central

    TIROTTA, EMANUELE; RANSOHOFF, RICHARD M.; LANE, THOMAS E.

    2016-01-01

    Infiltration of activated lymphocytes into the central nervous system (CNS) is potentially harmful by damaging resident cells through release of cytokines. Among these is IFN-γ that is secreted by activated natural killer (NK) cells and T lymphocytes and can exert a cytotoxic effect on resident glial populations including oligodendrocytes. Here we show that treatment of mouse oligodendrocyte progenitor cell (OPC)-enriched cultures with IFN-γ resulted in a dose-dependent increase in apoptosis. IFN-γ-induced apoptosis is mediated, in part, through induction of the CXC chemokine ligand 10 (CXCL10; IP-10) from cultured OPCs. Treatment of OPCs with CXCL10 resulted in cell death in a concentration-dependent manner and IFN-γ-treatment of CXCL10−/− OPCs resulted in >50% reduction in cell death. Further, treatment of CXCR3−/− OPC cultures with either IFN-γ or CXCL10 resulted in reduced cell death supporting an important role for CXCL10 signaling in IFN-γ-mediated OPC apoptosis. Data is also provided demonstrating that signaling through CXCR2 protects either IFN-γ or CXCL10-treated OPC cultures from apoptosis and this effect is abolished in CXCR2−/− OPCs. CXCR2-mediated protection from apoptosis is associated with impaired cleavage of caspase 3 and elevated expression of the anti-apoptotic protein Bcl-2. These findings demonstrate a previously unappreciated role for CXCL10 in contributing to neuropathology by promoting oligodendrocyte apoptosis and emphasize the potential relevance in targeting CXCL10 in treating human demyelinating diseases including multiple sclerosis (MS). PMID:21656856

  20. RAS signaling promotes resistance to JAK inhibitors by suppressing BAD-mediated apoptosis.

    PubMed

    Winter, Peter S; Sarosiek, Kristopher A; Lin, Kevin H; Meggendorfer, Manja; Schnittger, Susanne; Letai, Anthony; Wood, Kris C

    2014-12-23

    Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2(V617F) mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2(V617F) hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways [mediated by the kinases AKT and ERK (extracellular signal-regulated kinase)] renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2(V617F) cells, a JAK2-mediated inactivating phosphorylation of the proapoptotic protein BAD [B cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the prosurvival protein BCL-XL (BCL-2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. In patients with MPNs, activating mutations in RAS co-occur with the JAK2(V617F) mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance.

  1. The carboxyl terminal tyrosine 417 residue of NOK has an autoinhibitory effect on NOK-mediated signaling transductions

    SciTech Connect

    Li Yinghua; Zhong Shan; Rong Zhili; Ren Yongming; Li Zhiyong; Zhang Shuping; Chang Zhijie; Liu Li . E-mail: Liu_Li@mail.tsinghua.edu.cn

    2007-05-04

    Receptor protein tyrosine kinases (RPTKs) are essential mediators of cell growth, differentiation, migration, and metabolism. Recently, a novel RPTK named NOK has been cloned and characterized. In current study, we investigated the role of the carboxyl terminal tyrosine 417 residue of NOK in the activations of different signaling pathways. A single tyrosine to phenylalanine point mutation at Y417 site (Y417 F) not only dramatically enhanced the NOK-induced activation of extracellular signal-regulated kinase (ERK), but also markedly promoted the NOK-mediated activation of both signal transducer and activator of transcription 1 and 3 (STAT1 and 3). Moreover, the proliferation potential of NIH3T3-NOK (Y417F) stable cells were significantly elevated as compared with that of NIH3T3-NOK. Overall, our results demonstrate that the tyrosine Y417 residue at the carboxyl tail of NOK exhibits an autoinhibitory role in NOK-mediated signaling transductions.

  2. 7 CFR 1b.3 - Categorical exclusions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false Categorical exclusions. 1b.3 Section 1b.3 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.3 Categorical exclusions... individual or cumulative effect on the human environment and are excluded from the preparation...

  3. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 1 2013-01-01 2013-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of... environment for present and future generations. (b) Each USDA agency is responsible for compliance with...

  4. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of... environment for present and future generations. (b) Each USDA agency is responsible for compliance with...

  5. 7 CFR 1b.3 - Categorical exclusions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Categorical exclusions. 1b.3 Section 1b.3 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.3 Categorical exclusions... individual or cumulative effect on the human environment and are excluded from the preparation...

  6. 18 CFR 1b.6 - Preliminary investigations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Preliminary investigations. 1b.6 Section 1b.6 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.6 Preliminary investigations....

  7. 18 CFR 1b.14 - Subpoenas.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Subpoenas. 1b.14 Section 1b.14 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.14 Subpoenas. (a) Service of a...

  8. 18 CFR 1b.21 - Enforcement hotline.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Enforcement hotline. 1b.21 Section 1b.21 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.21 Enforcement hotline. (a)...

  9. 18 CFR 1b.12 - Transcripts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any,...

  10. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  11. 18 CFR 1b.2 - Scope.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Scope. 1b.2 Section 1b.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.2 Scope. This part applies to...

  12. 18 CFR 1b.2 - Scope.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Scope. 1b.2 Section 1b.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.2 Scope. This part applies to...

  13. 18 CFR 1b.12 - Transcripts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any,...

  14. 18 CFR 1b.6 - Preliminary investigations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Preliminary investigations. 1b.6 Section 1b.6 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.6 Preliminary investigations....

  15. 18 CFR 1b.6 - Preliminary investigations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Preliminary investigations. 1b.6 Section 1b.6 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.6 Preliminary investigations....

  16. 18 CFR 1b.2 - Scope.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Scope. 1b.2 Section 1b.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.2 Scope. This part applies to...

  17. 18 CFR 1b.21 - Enforcement hotline.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Enforcement hotline. 1b.21 Section 1b.21 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.21 Enforcement hotline. (a)...

  18. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  19. 18 CFR 1b.12 - Transcripts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any,...

  20. 18 CFR 1b.14 - Subpoenas.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Subpoenas. 1b.14 Section 1b.14 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.14 Subpoenas. (a) Service of a...

  1. 18 CFR 1b.21 - Enforcement hotline.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Enforcement hotline. 1b.21 Section 1b.21 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.21 Enforcement hotline. (a)...

  2. 18 CFR 1b.12 - Transcripts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any,...

  3. 18 CFR 1b.2 - Scope.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Scope. 1b.2 Section 1b.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.2 Scope. This part applies to...

  4. 18 CFR 1b.21 - Enforcement hotline.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Enforcement hotline. 1b.21 Section 1b.21 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.21 Enforcement hotline. (a)...

  5. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  6. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  7. 18 CFR 1b.14 - Subpoenas.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Subpoenas. 1b.14 Section 1b.14 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.14 Subpoenas. (a) Service of a...

  8. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  9. 18 CFR 1b.14 - Subpoenas.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Subpoenas. 1b.14 Section 1b.14 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.14 Subpoenas. (a) Service of a...

  10. 18 CFR 1b.6 - Preliminary investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Preliminary investigations. 1b.6 Section 1b.6 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.6 Preliminary investigations....

  11. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  12. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  13. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  14. 18 CFR 1b.6 - Preliminary investigations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Preliminary investigations. 1b.6 Section 1b.6 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.6 Preliminary investigations....

  15. 18 CFR 1b.12 - Transcripts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any,...

  16. 18 CFR 1b.14 - Subpoenas.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Subpoenas. 1b.14 Section 1b.14 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.14 Subpoenas. (a) Service of a...

  17. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  18. 18 CFR 1b.2 - Scope.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Scope. 1b.2 Section 1b.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.2 Scope. This part applies to...

  19. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  20. Association of transcription factor gene LMX1B with autism.

    PubMed

    Thanseem, Ismail; Nakamura, Kazuhiko; Anitha, Ayyappan; Suda, Shiro; Yamada, Kazuo; Iwayama, Yoshimi; Toyota, Tomoko; Tsujii, Masatsugu; Iwata, Yasuhide; Suzuki, Katsuaki; Matsuzaki, Hideo; Iwata, Keiko; Sugiyama, Toshiro; Yoshikawa, Takeo; Mori, Norio

    2011-01-01

    Multiple lines of evidence suggest a serotoninergic dysfunction in autism. The role of LMX1B in the development and maintenance of serotoninergic neurons is well known. In order to examine the role, if any, of LMX1B with autism pathophysiology, a trio-based SNP association study using 252 family samples from the AGRE was performed. Using pair-wise tagging method, 24 SNPs were selected from the HapMap data, based on their location and minor allele frequency. Two SNPs (rs10732392 and rs12336217) showed moderate association with autism with p values 0.018 and 0.022 respectively in transmission disequilibrium test. The haplotype AGCGTG also showed significant association (p = 0.008). Further, LMX1B mRNA expressions were studied in the postmortem brain tissues of autism subjects and healthy controls samples. LMX1B transcripts was found to be significantly lower in the anterior cingulate gyrus region of autism patients compared with controls (p = 0.049). Our study suggests a possible role of LMX1B in the pathophysiology of autism. Based on previous reports, it is likely to be mediated through a seretoninergic mechanism. This is the first report on the association of LMX1B with autism, though it should be viewed with some caution considering the modest associations we report.

  1. Association of Transcription Factor Gene LMX1B with Autism

    PubMed Central

    Yamada, Kazuo; Iwayama, Yoshimi; Toyota, Tomoko; Tsujii, Masatsugu; Iwata, Yasuhide; Suzuki, Katsuaki; Matsuzaki, Hideo; Iwata, Keiko; Sugiyama, Toshiro; Yoshikawa, Takeo; Mori, Norio

    2011-01-01

    Multiple lines of evidence suggest a serotoninergic dysfunction in autism. The role of LMX1B in the development and maintenance of serotoninergic neurons is well known. In order to examine the role, if any, of LMX1B with autism pathophysiology, a trio-based SNP association study using 252 family samples from the AGRE was performed. Using pair-wise tagging method, 24 SNPs were selected from the HapMap data, based on their location and minor allele frequency. Two SNPs (rs10732392 and rs12336217) showed moderate association with autism with p values 0.018 and 0.022 respectively in transmission disequilibrium test. The haplotype AGCGTG also showed significant association (p = 0.008). Further, LMX1B mRNA expressions were studied in the postmortem brain tissues of autism subjects and healthy controls samples. LMX1B transcripts was found to be significantly lower in the anterior cingulate gyrus region of autism patients compared with controls (p = 0.049). Our study suggests a possible role of LMX1B in the pathophysiology of autism. Based on previous reports, it is likely to be mediated through a seretoninergic mechanism. This is the first report on the association of LMX1B with autism, though it should be viewed with some caution considering the modest associations we report. PMID:21901133

  2. Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

    PubMed

    Ito, Naoki; Ruegg, Urs T; Kudo, Akira; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

    2013-01-01

    Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

  3. Aripiprazole has functionally selective actions at dopamine D2 receptor-mediated signaling pathways.

    PubMed

    Urban, Jonathan D; Vargas, Gabriel A; von Zastrow, Mark; Mailman, Richard B

    2007-01-01

    Aripiprazole is a unique atypical antipsychotic drug with an excellent side-effect profile presumed, in part, to be due to lack of typical D(2) dopamine receptor antagonist properties. Whether aripiprazole is a typical D(2) partial agonist, or a functionally selective D(2) ligand, remains controversial (eg D(2)-mediated inhibition of adenylate cyclase is system dependent; aripiprazole antagonizes D(2) receptor-mediated G-protein-coupled inwardly rectifying potassium channels and guanosine triphosphate nucleotide (GTP)gammaS coupling). The current study examined the D(2L) receptor binding properties of aripiprazole, as well as the effects of the drug on three downstream D(2) receptor-mediated functional effectors: mitogen-activated protein kinase (MAPK) phosphorylation, potentiation of arachidonic acid (AA) release, and D(2) receptor internalization. Unlike quinpirole (a full D(2) agonist) or (-)3PPP (S(-)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride, a D(2) partial agonist), the apparent D(2) affinity of aripiprazole was not decreased significantly by GTP. Moreover, full or partial agonists are expected to have Hill slopes <1.0, yet that of aripiprazole was significantly >1.0. Whereas aripiprazole partially activated both the MAPK and AA pathways, its potency vs MAPK phosphorylation was much lower relative to potencies in assays either of AA release or inhibition of cyclic adenosine 3',5'-cyclic monophosphate accumulation. In addition, unlike typical agonists, neither aripiprazole nor (-)3PPP produced significant internalization of the D(2L) receptor. These data are clear evidence that aripiprazole affects D(2L)-mediated signaling pathways in a differential manner. The results are consistent with the hypothesis that aripiprazole is a functionally selective D(2) ligand rather than a simple partial agonist. Such data may be useful in understanding the novel clinical actions of this drug.

  4. Orexin/hypocretin receptor 1 signaling mediates Pavlovian cue-food conditioning and extinction.

    PubMed

    Keefer, Sara E; Cole, Sindy; Petrovich, Gorica D

    2016-08-01

    Learned food cues can drive feeding in the absence of hunger, and orexin/hypocretin signaling is necessary for this type of overeating. The current study examined whether orexin also mediates cue-food learning during the acquisition and extinction of these associations. In Experiment 1, rats underwent two sessions of Pavlovian appetitive conditioning, consisting of tone-food presentations. Prior to each session, rats received either the orexin 1 receptor antagonist SB-334867 (SB) or vehicle systemically. SB treatment did not affect conditioned responses during the first conditioning session, measured as food cup behavior during the tone and latency to approach the food cup after the tone onset, compared to the vehicle group. During the second conditioning session, SB treatment attenuated learning. All groups that received SB, prior to either the first or second conditioning session, displayed significantly less food cup behavior and had longer latencies to approach the food cup after tone onset compared to the vehicle group. These findings suggest orexin signaling at the 1 receptor mediates the consolidation and recall of cue-food acquisition. In Experiment 2, another group of rats underwent tone-food conditioning sessions (drug free), followed by two extinction sessions under either SB or vehicle treatment. Similar to Experiment 1, SB did not affect conditioned responses during the first session. During the second extinction session, the group that received SB prior to the first extinction session, but vehicle prior to the second, expressed conditioned food cup responses longer after tone offset, when the pellets were previously delivered during conditioning, and maintained shorter latencies to approach the food cup compared to the other groups. The persistence of these conditioned behaviors indicates impairment in extinction consolidation due to SB treatment during the first extinction session. Together, these results demonstrate an important role for orexin

  5. STAT3 interrupts ATR-Chk1 signaling to allow oncovirus-mediated cell proliferation.

    PubMed

    Koganti, Siva; Hui-Yuen, Joyce; McAllister, Shane; Gardner, Benjamin; Grasser, Friedrich; Palendira, Umaimainthan; Tangye, Stuart G; Freeman, Alexandra F; Bhaduri-McIntosh, Sumita

    2014-04-01

    DNA damage response (DDR) is a signaling network that senses DNA damage and activates response pathways to coordinate cell-cycle progression and DNA repair. Thus, DDR is critical for maintenance of genome stability, and presents a powerful defense against tumorigenesis. Therefore, to drive cell-proliferation and transformation, viral and cellular oncogenes need to circumvent DDR-induced cell-cycle checkpoints. Unlike in hereditary cancers, mechanisms that attenuate DDR and disrupt cell-cycle checkpoints in sporadic cancers are not well understood. Using Epstein-Barr virus (EBV) as a source of oncogenes, we have previously shown that EBV-driven cell proliferation requires the cellular transcription factor STAT3. EBV infection is rapidly followed by activation and increased expression of STAT3, which mediates relaxation of the intra-S phase cell-cycle checkpoint; this facilitates viral oncogene-driven cell proliferation. We now show that replication stress-associated DNA damage, which results from EBV infection, is detected by DDR. However, signaling downstream of ATR is impaired by STAT3, leading to relaxation of the intra-S phase checkpoint. We find that STAT3 interrupts ATR-to-Chk1 signaling by promoting loss of Claspin, a protein that assists ATR to phosphorylate Chk1. This loss of Claspin which ultimately facilitates cell proliferation is mediated by caspase 7, a protein that typically promotes cell death. Our findings demonstrate how STAT3, which is constitutively active in many human cancers, suppresses DDR, fundamental to tumorigenesis. This newly recognized role for STAT3 in attenuation of DDR, discovered in the context of EBV infection, is of broad interest as the biology of cell proliferation is central to both health and disease.

  6. Orexin/hypocretin receptor 1 signaling mediates Pavlovian cue-food conditioning and extinction.

    PubMed

    Keefer, Sara E; Cole, Sindy; Petrovich, Gorica D

    2016-08-01

    Learned food cues can drive feeding in the absence of hunger, and orexin/hypocretin signaling is necessary for this type of overeating. The current study examined whether orexin also mediates cue-food learning during the acquisition and extinction of these associations. In Experiment 1, rats underwent two sessions of Pavlovian appetitive conditioning, consisting of tone-food presentations. Prior to each session, rats received either the orexin 1 receptor antagonist SB-334867 (SB) or vehicle systemically. SB treatment did not affect conditioned responses during the first conditioning session, measured as food cup behavior during the tone and latency to approach the food cup after the tone onset, compared to the vehicle group. During the second conditioning session, SB treatment attenuated learning. All groups that received SB, prior to either the first or second conditioning session, displayed significantly less food cup behavior and had longer latencies to approach the food cup after tone onset compared to the vehicle group. These findings suggest orexin signaling at the 1 receptor mediates the consolidation and recall of cue-food acquisition. In Experiment 2, another group of rats underwent tone-food conditioning sessions (drug free), followed by two extinction sessions under either SB or vehicle treatment. Similar to Experiment 1, SB did not affect conditioned responses during the first session. During the second extinction session, the group that received SB prior to the first extinction session, but vehicle prior to the second, expressed conditioned food cup responses longer after tone offset, when the pellets were previously delivered during conditioning, and maintained shorter latencies to approach the food cup compared to the other groups. The persistence of these conditioned behaviors indicates impairment in extinction consolidation due to SB treatment during the first extinction session. Together, these results demonstrate an important role for orexin

  7. Central mechanisms mediating the hypophagic effects of oleoylethanolamide and N-acylphosphatidylethanolamines: different lipid signals?

    PubMed Central

    Romano, Adele; Tempesta, Bianca; Provensi, Gustavo; Passani, Maria B.; Gaetani, Silvana

    2015-01-01

    The spread of “obesity epidemic” and the poor efficacy of many anti-obesity therapies in the long-term highlight the need to develop novel efficacious therapy. This necessity stimulates a large research effort to find novel mechanisms controlling feeding and energy balance. Among these mechanisms a great deal of attention has been attracted by a family of phospholipid-derived signaling molecules that play an important role in the regulation of food-intake. They include N-acylethanolamines (NAEs) and N-acylphosphatidylethanolamines (NAPEs). NAPEs have been considered for a long time simply as phospholipid precursors of the lipid mediator NAEs, but increasing body of evidence suggest a role in many physiological processes including the regulation of feeding behavior. Several observations demonstrated that among NAEs, oleoylethanolamide (OEA) acts as a satiety signal, which is generated in the intestine, upon the ingestion of fat, and signals to the central nervous system. At this level different neuronal pathways, including oxytocinergic, noradrenergic, and histaminergic neurons, seem to mediate its hypophagic action. Similarly to NAEs, NAPE (with particular reference to the N16:0 species) levels were shown to be regulated by the fed state and this finding was initially interpreted as fluctuations of NAE precursors. However, the observation that exogenously administered NAPEs are able to inhibit food intake, not only in normal rats and mice but also in mice lacking the enzyme that converts NAPEs into NAEs, supported the hypothesis of a role of NAPE in the regulation of feeding behavior. Indirect observations suggest that the hypophagic action of NAPEs might involve central mechanisms, although the molecular target remains unknown. The present paper reviews the role that OEA and NAPEs play in the mechanisms that control food intake, further supporting this group of phospholipids as optimal candidate for the development of novel anti-obesity treatments. PMID

  8. Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

    PubMed

    Liang, Hai Po H; Kerschen, Edward J; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; Jia, Shuang; Hessner, Martin J; Toso, Raffaella; Rezaie, Alireza R; Fernández, José A; Camire, Rodney M; Ruf, Wolfram; Griffin, John H; Weiler, Hartmut

    2015-11-19

    The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

  9. Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

    PubMed Central

    Mühlhaus, Jessica; Dinter, Juliane; Nürnberg, Daniela; Rehders, Maren; Depke, Maren; Golchert, Janine; Homuth, Georg; Yi, Chun-Xia; Morin, Silke; Köhrle, Josef; Brix, Klaudia; Tschöp, Matthias; Kleinau, Gunnar; Biebermann, Heike

    2014-01-01

    The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs. PMID:25391046

  10. UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum*

    PubMed Central

    Lee, Dukgyu; Kraus, Allison; Prins, Daniel; Groenendyk, Jody; Aubry, Isabelle; Liu, Wen-Xin; Li, Hao-Dong; Julien, Olivier; Touret, Nicolas; Sykes, Brian D.; Tremblay, Michel L.; Michalak, Marek

    2015-01-01

    Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism. PMID:25586181

  11. Extracellular ATP signaling via P2X(4) receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2012-09-01

    Prechondrogenic condensation is the most critical process in skeletal patterning. A previous study demonstrated that ATP oscillations driven by Ca(2+) oscillations play a critical role in prechondrogenic condensation by inducing oscillatory secretion. However, it remains unknown what mechanisms initiate the Ca(2+)-driven ATP oscillations, mediate the link between Ca(2+) and ATP oscillations, and then result in oscillatory secretion in chondrogenesis. This study has shown that extracellular ATP signaling was required for both ATP oscillations and prechondrogenic condensation. Among P2 receptors, the P2X(4) receptor revealed the strongest expression level and mediated ATP oscillations in chondrogenesis. Moreover, blockage of P2X(4) activity abrogated not only chondrogenic differentiation but also prechondrogenic condensation. In addition, both ATP oscillations and secretion activity depended on cAMP/PKA signaling but not on K(ATP) channel activity and PKC or PKG signaling. This study proposes that Ca(2+)-driven ATP oscillations essential for prechondrogenic condensation is initiated by extracellular ATP signaling via P2X(4) receptor and is mediated by cAMP/PKA signaling and that cAMP/PKA signaling induces oscillatory secretion to underlie prechondrogenic condensation, in cooperation with Ca(2+) and ATP oscillations.

  12. Adenosine signaling inhibits CIITA-mediated MHC class II transactivation in lung fibroblast cells.

    PubMed

    Fang, Mingming; Xia, Jun; Wu, Xiaoyan; Kong, Hui; Wang, Hong; Xie, Weiping; Xu, Yong

    2013-08-01

    Efficient antigen presentation by major histocompatibility complex (MHC) molecules represents a critical process in adaptive immunity. Class II transactivator (CIITA) is considered the master regulator of MHC class II (MHC II) transcription. Previously, we have shown that CIITA expression is upregulated in smooth muscle cells deficient in A2b adenosine receptor. Here, we report that treatment with the adenosine receptor agonist adenosine-5'N-ethylcarboxamide (NECA) attenuated MHC II transcription in lung fibro-blast cells as a result of CIITA repression. Further analysis revealed that NECA preferentially abrogated CIITA transcription through promoters III and IV. Blockade with a selective A2b receptor antagonist MRS-1754 restored CIITA-dependent MHC II transactivation. Forskolin, an adenylyl cyclase activator, achieved the same effect as NECA. A2b signaling repressed CIITA transcription by altering histone modifications and recruitment of key factors on the CIITA promoters in a STAT1-dependent manner. MRS-1754 blocked the antagonism of transforming growth factor beta (TGF-β) in CIITA induction by interferon gamma (IFN-γ), alluding to a potential dialogue between TGF-β and adenosine signaling pathways. Finally, A2b signaling attenuated STAT1 phosphorylation and stimulated TGF-β synthesis. In conclusion, we have identified an adenosine-A2b receptor-adenylyl cyclase axis that influences CIITA-mediated MHC II transactivation in lung fibroblast cells and as such have provided invaluable insights into the development of novel immune-modulatory strategies.

  13. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    PubMed

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs. PMID:27499021

  14. Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation.

    PubMed

    Wang, Jirong R; Wang, Chaojun J; Xu, Chengyun Y; Wu, Xiaokai K; Hong, Dun; Shi, Wei; Gong, Ying; Chen, Haixiao X; Long, Fanxin; Wu, Ximei M

    2016-03-01

    Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation.

  15. Type 3 adenylyl cyclase: a key enzyme mediating the cAMP signaling in neuronal cilia

    PubMed Central

    Qiu, Liyan; LeBel, Robert P; Storm, Daniel R; Chen, Xuanmao

    2016-01-01

    Cilia are rigid, centriole-derived, microtubule-based organelles present in a majority of vertebrate cells including neurons. They are considered the cellular “antennae” attuned for detecting a range of extracellular signals including photons, odorants, morphogens, hormones and mechanical forces. The ciliary microenvironment is distinct from most actin-based subcellular structures such as microvilli or synapses. In the nervous system, there is no evidence that neuronal cilia process any synaptic structure. Apparently, the structural features of neuronal cilia do not allow them to harbor any synaptic connections. Nevertheless, a large number of G protein-coupled receptors (GPCRs) including odorant receptors, rhodopsin, Smoothened, and type 6 serotonin receptor are found in cilia, suggesting that these tiny processes largely depend on metabotropic receptors and their tuned signals to impact neuronal functions. The type 3 adenylyl cyclase (AC3), widely known as a cilia marker, is highly and predominantly expressed in olfactory sensory cilia and primary cilia throughout the brain. We discovered that ablation of AC3 in mice leads to pleiotropic phenotypes including anosmia, failure to detect mechanical stimulation of airflow, cognitive deficit, obesity, and depression-like behaviors. Multiple lines of human genetic evidence also demonstrate that AC3 is associated with obesity, major depressive disorder (MDD), sarcoidosis, and infertility, underscoring its functional importance. Here we review recent progress on AC3, a key enzyme mediating the cAMP signaling in neuronal cilia. PMID:27785336

  16. A Systems Level Analysis of Vasopressin-mediated Signaling Networks in Kidney Distal Convoluted Tubule Cells

    PubMed Central

    Cheng, Lei; Wu, Qi; Kortenoeven, Marleen L. A.; Pisitkun, Trairak; Fenton, Robert A.

    2015-01-01

    The kidney distal convoluted tubule (DCT) plays an essential role in maintaining body sodium balance and blood pressure. The major sodium reabsorption pathway in the DCT is the thiazide-sensitive NaCl cotransporter (NCC), whose functions can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. Here we use a systems biology approach centered on stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells to map global changes in protein phosphorylation upon acute treatment with a VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). 6330 unique proteins, containing 12333 different phosphorylation sites were identified. 185 sites were altered in abundance following dDAVP. Basophilic motifs were preferential targets for upregulated sites upon dDAVP stimulation, whereas proline-directed motifs were prominent for downregulated sites. Kinase prediction indicated that dDAVP increased AGC and CAMK kinase families’ activities and decreased activity of CDK and MAPK families. Network analysis implicated phosphatidylinositol-4,5-bisphosphate 3-kinase or CAMKK dependent pathways in VP-mediated signaling; pharmacological inhibition of which significantly reduced dDAVP induced increases in phosphorylated NCC at an activating site. In conclusion, this study identifies unique VP signaling cascades in DCT cells that may be important for regulating blood pressure. PMID:26239621

  17. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    PubMed

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

  18. TRIF mediates Toll-like receptor 5-induced signaling in intestinal epithelial cells.

    PubMed

    Choi, Yoon Jeong; Im, Eunok; Chung, Hyo Kyun; Pothoulakis, Charalabos; Rhee, Sang Hoon

    2010-11-26

    Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.

  19. Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells.

    PubMed

    Gonçalves, Vânia; Henriques, Andreia F A; Henriques, Andreia; Pereira, Joana F S; Pereira, Joana; Neves Costa, Ana; Moyer, Mary Pat; Moita, Luís Ferreira; Gama-Carvalho, Margarida; Matos, Paulo; Jordan, Peter

    2014-04-01

    The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3β, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.

  20. Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors.

    PubMed

    Wurch, T; Palmier, C; Colpaert, F C; Pauwels, P J

    1997-01-01

    1. The rabbit recombinant saphenous vein 5-hydroxytryptamine1B (r 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3':5'-cyclic monophosphate (cycle AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. 2. Intact C6-glial cells expressing rb HT1B receptors exhibited [3H]-5-carboxamidotryptamine (5-CT) binding sites with a Kd of 0.80 +/- 0.13 nM and a Bmax between 225 to 570 fmol mg-1 protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [3H]-5-CT or [3H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(-4 -pyridyl) benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the clones h 5-HT1B receptor site. 3. rb 5-HT1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT > 5-HT > zolmitriptan > naratriptan > rizatriptan > sumatriptan > R (+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r2 = 0.87; P < 0.002) with their potency at the cloned h 5-HT1B receptor subtype. 4. 2'-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-e-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5HT1B receptors; pKB values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated

  1. Endothelin-1/endothelin A receptor-mediated biased signaling is a new player in modulating human ovarian cancer cell tumorigenesis.

    PubMed

    Teoh, Jian-peng; Park, Kyoung-mi; Wang, Yongchao; Hu, Qiuping; Kim, Sangmi; Wu, Guangyu; Huang, Shuang; Maihle, Nita; Kim, Il-man

    2014-12-01

    The endothelin-1 (ET-1)/endothelin A receptor (ETAR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and β-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ETAR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ETAR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ETAR-targeted OC therapy, we investigated the role of other G protein subunits such as Gαs in the ETAR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ETAR axis is well-characterized, Gαs signaling inhibits ETAR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ETAR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ETAR-mediated β-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ETAR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/β-arrestin-mediated oncogenic pathways, to improve the targeting of the ETAR axis in OC.

  2. Isolation of Modulators of the Liver-Specific Organic Anion-Transporting Polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae)

    PubMed Central

    Roth, Megan; Araya, Juan J.; Timmermann, Barbara N.

    2011-01-01

    Organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver-specific transporters that mediate the uptake of a broad range of drugs into hepatocytes, including statins, antibiotics, and many anticancer drugs. Compounds that alter transport by one or both of these OATPs could potentially be used to target drugs to hepatocytes or improve the bioavailability of drugs that are cleared by the liver. In this study, we applied a bioassay-guided isolation approach to identify such compounds from the organic extract of Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for effects on OATP1B1- and OATP1B3-mediated transport of the model substrates estradiol-17β-glucuronide and estrone-3-sulfate. We isolated three compounds, ursolic acid, oleanolic acid, and 8-trans-p-coumaroyloxy-α-terpineol, which inhibited estradiol-17β-glucuronide uptake by OATP1B1 but not OATP1B3. In addition, a rare compound, quercetin 3-O-α-l-arabinopyranosyl(1→2) α-l-rhamnopyranoside, was identified that had distinct effects on each OATP. OATP1B1 was strongly inhibited, as was OATP1B3-mediated transport of estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate was stimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity for estrone-3-sulfate was increased (decreased Km), whereas the maximal rate of transport (Vmax) was significantly reduced. These results demonstrate a mechanism through which the hepatic uptake of drug OATP substrates could be stimulated. PMID:21846839

  3. Isolation of modulators of the liver-specific organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae).

    PubMed

    Roth, Megan; Araya, Juan J; Timmermann, Barbara N; Hagenbuch, Bruno

    2011-11-01

    Organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver-specific transporters that mediate the uptake of a broad range of drugs into hepatocytes, including statins, antibiotics, and many anticancer drugs. Compounds that alter transport by one or both of these OATPs could potentially be used to target drugs to hepatocytes or improve the bioavailability of drugs that are cleared by the liver. In this study, we applied a bioassay-guided isolation approach to identify such compounds from the organic extract of Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for effects on OATP1B1- and OATP1B3-mediated transport of the model substrates estradiol-17β-glucuronide and estrone-3-sulfate. We isolated three compounds, ursolic acid, oleanolic acid, and 8-trans-p-coumaroyloxy-α-terpineol, which inhibited estradiol-17β-glucuronide uptake by OATP1B1 but not OATP1B3. In addition, a rare compound, quercetin 3-O-α-l-arabinopyranosyl(1→2) α-L-rhamnopyranoside, was identified that had distinct effects on each OATP. OATP1B1 was strongly inhibited, as was OATP1B3-mediated transport of estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate was stimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity for estrone-3-sulfate was increased (decreased K(m)), whereas the maximal rate of transport (V(max)) was significantly reduced. These results demonstrate a mechanism through which the hepatic uptake of drug OATP substrates could be stimulated.

  4. TIMP-1 mediates TGF-β-dependent crosstalk between hepatic stellate and cancer cells via FAK signaling.

    PubMed

    Park, Sang-A; Kim, Min-Jin; Park, So-Yeon; Kim, Jung-Shin; Lim, Woosung; Nam, Jeong-Seok; Yhong Sheen, Yhun

    2015-01-01

    Transforming growth factor-β (TGF-β) signaling plays a key role in progression and metastasis of HCC. This study was undertaken to gain the proof of concept of a small-molecule inhibitor of TGF-β type I receptor kinase, EW-7197 as a potent anti-cancer therapy for HCC. We identified tissue inhibitors of metalloproteinases-1 (TIMP-1) as one of the secreted proteins of hepatic stellate cells (HSCs) and a key mediator of TGF-β-mediated crosstalk between HSCs and HCC cells. TGF-β signaling led to increased expression of TIMP-1, which activates focal adhesion kinase (FAK) signaling via its interaction with CD63. Inhibition of TGF-β signaling using EW-7197 significantly attenuated the progression and intrahepatic metastasis of HCC in an SK-HEP1-Luc orthotopic-xenograft mouse model. In addition, EW-7197 inhibited TGF-β-stimulated TIMP-1 secretion by HSCs as well as the TIMP-1-induced proliferation, motility, and survival of HCC cells. Further, EW-7197 interrupted TGF-β-mediated epithelial-to-mesenchymal transition and Akt signaling, leading to significant reductions in the motility and anchorage-independent growth of HCC cells. In conclusion, we found that TIMP-1 mediates TGF-β-regulated crosstalk between HSCs and HCC cells via FAK signaling. In addition, EW-7197 demonstrates potent in vivo anti-cancer therapeutic activity and may be a potential new anti-cancer drug of choice to treat patients with liver cancer. PMID:26549110

  5. Paracrine mediation of calcium signaling in human SK-N-MCIXC neuroepithelioma cells.

    PubMed

    Palmer, R K; Yule, D I; Shewach, D S; Williams, J A; Fisher, S K

    1996-07-01

    Paracrine-mediated Ca2+ signaling in SK-N-MCIXC neuroepithelioma cells was evaluated by means of two experimental paradigms. In the first, single SK-N-MCIXC cells were microinjected with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and cytoplasmic Ca2+ was monitored by fura 2 digital-imaging microfluorometry. In response to Ins(1,4,5)P3 or CaCl2, but not inositol 1,3,4-trisphosphate, an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) was observed in injected cells and also in neighboring cells. The direction of intercellular propagation of Ca2+ signals was influenced by the presence of a flow in the extracellular medium and occurred in the absence of any detectable gap-junctional communication. The P2 purinoceptor antagonist suramin, but not antagonists of other phosphoinositide-linked receptors, blocked cell-to-cell Ca2+ signaling initiated by microinjections of Ins(1,4,5)P3. In the second paradigm, conditioned medium (CM) obtained from monolayers of SK-N-MCIXC cells elicited increases in [Ca2+]i when reapplied to cells on coverslips. The Ca(2+)-mobilizing activity of CM was reversibly antagonized by suramin and abolished by pretreatment with apyrase. The presence of nucleotide di- and triphosphates in CM was confirmed by high-performance liquid chromatography. We conclude that SK-N-MCIXC cells release nucleotides that then activate specific receptors on neighboring cells. A rise in [Ca2+]i in these cells, and subsequent additional release of nucleotides, serves to further the propagation of intercellular Ca2+ signals.

  6. 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

  7. Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.

    PubMed

    Le Cras, Timothy D; Acciani, Thomas H; Mushaben, Elizabeth M; Kramer, Elizabeth L; Pastura, Patricia A; Hardie, William D; Korfhagen, Thomas R; Sivaprasad, Umasundari; Ericksen, Mark; Gibson, Aaron M; Holtzman, Michael J; Whitsett, Jeffrey A; Hershey, Gurjit K Khurana

    2011-03-01

    Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.

  8. Protein phosphatases type 2A mediate tuberization signaling in Solanum tuberosum L. leaves.

    PubMed

    País, Silvia Marina; García, María Noelia Muñiz; Téllez-Iñón, María Teresa; Capiati, Daniela Andrea

    2010-06-01

    Tuber formation in potato (Solanum tuberosum L.) is regulated by hormonal and environmental signals that are thought to be integrated in the leaves. The molecular mechanisms that mediate the responses to tuberization-related signals in leaves remain largely unknown. In this study we analyzed the roles of protein phosphatase type 2A catalytic subunits (PP2Ac) in the leaf responses to conditions that affect tuberization. The responses were monitored by analyzing the expression of the "tuber-specific" genes Patatin and Pin2, which are induced in tubers and leaves during tuber induction. Experiments using PP2A inhibitors, together with PP2Ac expression profiles under conditions that affect tuberization indicate that high sucrose/nitrogen ratio, which promotes tuber formation, increases the transcript levels of Patatin and Pin2, by increasing the activity of PP2As without affecting PP2Ac mRNA or protein levels. Gibberellic acid (GA), a negative regulator of tuberization, down-regulates the transcription of catalytic subunits of PP2As from the subfamily I and decreases their enzyme levels. In addition, GA inhibits the expression of Patatin and Pin2 possibly by a PP2A-independent mechanism. PP2Ac down-regulation by GA may inhibit tuberization signaling downstream of the inductive effects of high sucrose/nitrogen ratio. These results are consistent with the hypothesis that PP2As of the subfamily I may positively modulate the signaling pathways that lead to the transcriptional activation of "tuber-specific" genes in leaves, and act as molecular switches regulated by both positive and negative modulators of tuberization. PMID:20358221

  9. Emergence of the A20/ABIN-mediated inhibition of NF-κB signaling via modifying the ubiquitinated proteins in a basal chordate.

    PubMed

    Yuan, Shaochun; Dong, Xiangru; Tao, Xin; Xu, Liqun; Ruan, Jie; Peng, Jian; Xu, Anlong

    2014-05-01

    In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling.

  10. Emergence of the A20/ABIN-mediated inhibition of NF-κB signaling via modifying the ubiquitinated proteins in a basal chordate.

    PubMed

    Yuan, Shaochun; Dong, Xiangru; Tao, Xin; Xu, Liqun; Ruan, Jie; Peng, Jian; Xu, Anlong

    2014-05-01

    In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling. PMID:24753567

  11. Salt and gene expression: evidence for [Na+]i/[K+]i-mediated signaling pathways.

    PubMed

    Orlov, Sergei N; Hamet, Pavel

    2015-03-01

    Our review focuses on the recent data showing that gene transcription and translation are under the control of signaling pathways triggered by modulation of the intracellular sodium/potassium ratio ([Na+]i/[K+]i). Side-by-side with sensing of osmolality elevation by tonicity enhancer-binding protein (TonEBP, NFAT5), [Na+]i/[K+]i-mediated excitation-transcription coupling may contribute to the transcriptomic changes evoked by high salt consumption. This novel mechanism includes the sensing of heightened Na+ concentration in the plasma, interstitial, and cerebrospinal fluids via augmented Na+ influx in the endothelium, immune system cells, and the subfornical organ, respectively. In these cells, [Na+]i/[K+]i ratio elevation, triggered by augmented Na+ influx, is further potentiated by increased production of endogenous Na+,K+-ATPase inhibitors documented in salt-sensitive hypertension. PMID:25479826

  12. Inflammatory mediators and insulin resistance in obesity: role of nuclear receptor signaling in macrophages.

    PubMed

    Fuentes, Lucía; Roszer, Tamás; Ricote, Mercedes

    2010-01-01

    Visceral obesity is coupled to a general low-grade chronic inflammatory state characterized by macrophage activation and inflammatory cytokine production, leading to insulin resistance (IR). The balance between proinflammatory M1 and antiinflammatory M2 macrophage phenotypes within visceral adipose tissue appears to be crucially involved in the development of obesity-associated IR and consequent metabolic abnormalities. The ligand-dependent transcription factors peroxisome proliferator activated receptors (PPARs) have recently been implicated in the determination of the M1/M2 phenotype. Liver X receptors (LXRs), which form another subgroup of the nuclear receptor superfamily, are also important regulators of proinflammatory cytokine production in macrophages. Disregulation of macrophage-mediated inflammation by PPARs and LXRs therefore underlies the development of IR. This review summarizes the role of PPAR and LXR signaling in macrophages and current knowledge about the impact of these actions in the manifestation of IR and obesity comorbidities such as liver steatosis and diabetic osteopenia.

  13. Lactate-mediated glia-neuronal signalling in the mammalian brain

    NASA Astrophysics Data System (ADS)

    Tang, F.; Lane, S.; Korsak, A.; Paton, J. F. R.; Gourine, A. V.; Kasparov, S.; Teschemacher, A. G.

    2014-02-01

    Astrocytes produce and release L-lactate as a potential source of energy for neurons. Here we present evidence that L-lactate, independently of its caloric value, serves as an astrocytic signalling molecule in the locus coeruleus (LC). The LC is the principal source of norepinephrine to the frontal brain and thus one of the most influential modulatory centers of the brain. Optogenetically activated astrocytes release L-lactate, which excites LC neurons and triggers release of norepinephrine. Exogenous L-lactate within the physiologically relevant concentration range mimics these effects. L-lactate effects are concentration-dependent, stereo-selective, independent of L-lactate uptake into neurons and involve a cAMP-mediated step. In vivo injections of L-lactate in the LC evokes arousal similar to the excitatory transmitter, L-glutamate. Our results imply the existence of an unknown receptor for this ‘glio-transmitter’.

  14. Hormone-Mediated Intercellular Calcium Signalling in an Insect Salivary Gland Pathways and Mechanisms

    NASA Astrophysics Data System (ADS)

    Zimmermann, Bernhard; Walz, Bernd

    The salivary glands of the blowfly Calliphora vicina are a favourable preparation for investigations into spatio-temporal Ca 2+ dynamics in an intact miniorgan by using Ca 2+-sensitive indicator dyes and digital imaging techniques, including confocal microscopy, in combination with pharmacological approaches. The review summarizes the available data on the spatio-temporal patterns of the hormone-induced and IP 3-mediated Ca 2+ dynamics at both the intracellular and the intercellular level (intra- and intercellular Ca 2+ waves). The underlying signaling mechanisms are addressed, as well as the pathways of intercellular communication responsible for the complex spatio-temporal Ca 2+ dynamics. In addition, we review evidence for the exchange of Ca 2+ between IP 3 sensitive intracellular Ca 2+ stores and mitochondria including a modulatory effect of mitochondrial Ca 2+ uptake on the frequency of IP 3-induced Ca 2+ spiking.

  15. Lactate-mediated glia-neuronal signalling in the mammalian brain

    PubMed Central

    Tang, F.; Lane, S.; Korsak, A.; Paton, J. F. R.; Gourine, A. V.; Kasparov, S.; Teschemacher, A. G.

    2014-01-01

    Astrocytes produce and release L-lactate as a potential source of energy for neurons. Here we present evidence that L-lactate, independently of its caloric value, serves as an astrocytic signalling molecule in the locus coeruleus (LC). The LC is the principal source of norepinephrine to the frontal brain and thus one of the most influential modulatory centers of the brain. Optogenetically activated astrocytes release L-lactate, which excites LC neurons and triggers release of norepinephrine. Exogenous L-lactate within the physiologically relevant concentration range mimics these effects. L-lactate effects are concentration-dependent, stereo-selective, independent of L-lactate uptake into neurons and involve a cAMP-mediated step. In vivo injections of L-lactate in the LC evokes arousal similar to the excitatory transmitter, L-glutamate. Our results imply the existence of an unknown receptor for this ‘glio-transmitter’. PMID:24518663

  16. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

    PubMed

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  17. Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

    PubMed Central

    Wheeler, David S.; Underhill, Suzanne M.; Stolz, Donna B.; Murdoch, Geoffrey H.; Thiels, Edda; Romero, Guillermo; Amara, Susan G.

    2015-01-01

    Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action. PMID:26553986

  18. Extracellular ATP mediates Ca2+ sign