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Sample records for mediates extracellular signal-regulated

  1. 5-HT7 receptor-mediated fear conditioning and possible involvement of extracellular signal-regulated kinase.

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    Takeda, Kotaro; Tsuji, Minoru; Miyagawa, Kazuya; Takeda, Hiroshi

    2017-01-18

    Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The present study examined the involvement of extracellular signal-regulated kinase 1/2 (ERK) signaling on the serotonin (5-HT)7 receptor-mediated fear conditioning. Conditioning was performed in a trial in which a tone was followed by an electrical foot-shock. Context- and tone-dependent fear were examined in tests conducted 24 and 48h after conditioning, respectively. The selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,-tetrahydrobenzo(c,d)indol-2-(1H)-one (DR4004) (5mg/kg), when administered intraperitoneally (i.p.) immediately after conditioning, caused a significant decrease in both context- and tone-dependent fear responses (freezing behavior). A significant increase in ERK activity was observed in the amygdala of mice that displayed context- or tone-dependent fear responses, and these changes were also inhibited by the administration of DR4004 (5mg/kg, i.p.) immediately after conditioning. In contrast, the increase in hippocampal ERK activity in mice that displayed context-dependent fear responses was further enhanced by the administration of DR4004 (5mg/kg, i.p.). These results suggest that 5-HT7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory.

  2. NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

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    Kuek, Vincent; Yang, Zhifan; Chim, Shek Man; Zhu, Sipin; Xu, Huazi; Chow, Siu To; Tickner, Jennifer; Rosen, Vicki; Erber, Wendy; Li, Xiucheng; An, Qin; Qian, Yu; Xu, Jiake

    2016-01-01

    Angiogenesis plays an important role in bone development and remodeling and is mediated by a plethora of potential angiogenic factors. However, data regarding specific angiogenic factors that are secreted within the bone microenvironment to regulate osteoporosis is lacking. Here, we report that Nephronectin (NPNT), a member of the epidermal growth factor (EGF) repeat superfamily proteins and a homologue of EGFL6, is expressed in osteoblasts. Intriguingly, the gene expression of NPNT is reduced in the bone of C57BL/6J ovariectomised mice and in osteoporosis patients. In addition, the protein levels of NPNT and CD31 are also found to be reduced in the tibias of OVX mice. Exogenous addition of mouse recombinant NPNT on endothelial cells stimulates migration and tube-like structure formation in vitro. Furthermore, NPNT promotes angiogenesis in an ex vivo fetal mouse metatarsal angiogenesis assay. We show that NPNT stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated kinase (MAPK) in endothelial cells. Inhibition of ERK1/2 impaired NPNT-induced endothelial cell migration, tube-like structure formation and angiogenesis. Taken together, these results demonstrate that NPNT is a paracrine angiogenic factor and may play a role in pathological osteoporosis. This may lead to new targets for treatment of bone diseases and injuries. PMID:27782206

  3. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

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    Puustinen, Pietri; Junttila, Melissa R; Vanhatupa, Sari; Sablina, Anna A; Hector, Melissa E; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C; Westermarck, Jukka

    2009-04-01

    Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

  4. Thrombopoietin potentiates the protein-kinase-C-mediated activation of mitogen-activated protein kinase/ERK kinases and extracellular signal-regulated kinases in human platelets.

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    Ezumi, Y; Uchiyama, T; Takayama, H

    1998-12-15

    The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal-regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C (PKC), whereas TPO directly enhanced the PKC-dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen-activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf-1 and directly augmented the PKC-mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf-1. The MEK inhibitor PD098059 failed to affect not only thrombin-induced or phorbol ester-induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. ERKs and MEKs were located mainly in the detergent-soluble/non-cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.

  5. Follicle-stimulating hormone (FSH activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization

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    Crepieux Pascale

    2006-06-01

    Full Text Available Abstract Background The follicle-stimulating hormone receptor (FSH-R is a seven transmembrane spanning receptor (7TMR which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK. However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. Methods Human embryonic kidney (HEK 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319–418 dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. Results In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319–418 construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. Conclusion From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH.

  6. Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish.

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    Klausen, Christian; Booth, Morgan; Habibi, Hamid R; Chang, John P

    2008-08-01

    The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4beta-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4alpha-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.

  7. Phospholipase A(2) activation by poultry particulate matter is mediated through extracellular signal-regulated kinase in lung epithelial cells: regulation of interleukin-8 release.

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    Kotha, Sainath R; Piper, Melissa G; Patel, Rishi B; Sliman, Sean; Malireddy, Smitha; Zhao, Lingying; Baran, Christopher P; Nana-Sinkam, Patrick S; Wewers, Mark D; Romberger, Debra; Marsh, Clay B; Parinandi, Narasimham L

    2013-11-01

    The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

  8. Regulation of enhanced cerebrovascular expression of proinflammatory mediators in experimental subarachnoid hemorrhage via the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway

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    Maddahi Aida

    2012-12-01

    Full Text Available Abstract Background Subarachnoid hemorrhage (SAH is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function. Methods SAH was induced in rats by injection of 250 μl of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL-6, IL-1β, tumor necrosis factor α (TNFα, matrix metalloproteinase (MMP-9 and phosphorylated ERK1/2 (pERK1/2 levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed. Results Expression levels of IL-1β, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNFα in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126

  9. P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells.

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    Eun, So Young; Ko, Young Shin; Park, Sang Won; Chang, Ki Churl; Kim, Hye Jung

    2015-07-01

    Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

  10. Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor kappaB-mediated gene expression in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Lykke; Størling, J; Darville, M;

    2005-01-01

    The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta activates mitogen-activated protein kinases (MAPKs), including extra...... by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFkappaB activation in beta cells....

  11. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

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    Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  12. Involvement of extracellular signal-regulated kinase (ERK1/2)-p53-p21 axis in mediating neural stem/progenitor cell cycle arrest in co-morbid HIV-drug abuse exposure.

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    Malik, Shaily; Saha, Rinki; Seth, Pankaj

    2014-06-01

    Neurological complications in opioid abusing Human Immunodeficiency Virus-1 (HIV-1) patients suggest enhanced neurodegeneration as compared to non-drug abusing HIV-1 infected population. Neural precursor cells (NPCs), the multipotent cells of the mammalian brain, are susceptible to HIV-1 infection and as opiates also perturb their growth kinetics, detailed mechanistic studies for their co-morbid exposure are highly warranted. Using a well characterized in vitro model of human fetal brain-derived neural precursor cells, we investigated alterations in NPC properties at both acute and chronic durations. Chronic morphine and Tat treatment attenuated proliferation in NPCs, with cells stalled at G1-phase of the cell cycle. Furthermore HIV-Tat and morphine exposure increased activation of extracellular signal-regulated kinase-1/2 (ERK1/2), enhanced levels of p53 and p21, and decreased cyclin D1 and Akt levels in NPCs. Regulated by ERK1/2 and p53, p21 was found to be indispensible for Tat and morphine mediated cell cycle arrest. Our study elaborates on the cellular and molecular machinery in NPCs and provides significant mechanistic details into HIV-drug abuse co-morbidity that may have far reaching clinical consequences both in pediatric as well as adult neuroAIDS.

  13. The Hinge-Helix 1 Region of Peroxisome Proliferator-Activated Receptor γ1 (PPARγ1) Mediates Interaction with Extracellular Signal-Regulated Kinase 5 and PPARγ1 Transcriptional Activation: Involvement in Flow-Induced PPARγ Activation in Endothelial Cells

    OpenAIRE

    Akaike, Masashi; Che, Wenyi; Marmarosh, Nicole-Lerner; Ohta, Shinsuke; Osawa, Masaki; Ding,Bo; Berk, Bradford C.; Yan, Chen; Abe, Jun-ichi

    2004-01-01

    Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPARγ have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPARγ activity. We found that activation of ERK5 induced PPARγ1 activation in endothelial cells (ECs). However, we could not detect PPARγ p...

  14. Intracellular transactivation of epidermal growth factor receptor by α1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells.

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    Ulu, Nadir; Henning, Robert H; Guner, Sahika; Zoto, Teuta; Duman-Dalkilic, Basak; Duin, Marry; Gurdal, Hakan

    2013-10-01

    Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N(1)-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.

  15. Brain-derived neurotrophic factor activation of extracellular signal-regulated kinase is autonomous from the dominant extrasynaptic NMDA receptor extracellular signal-regulated kinase shutoff pathway.

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    Mulholland, P J; Luong, N T; Woodward, J J; Chandler, L J

    2008-01-24

    NMDA receptors bidirectionally modulate extracellular signal-regulated kinase (ERK) through the coupling of synaptic NMDA receptors to an ERK activation pathway that is opposed by a dominant ERK shutoff pathway thought to be coupled to extrasynaptic NMDA receptors. In the present study, synaptic NMDA receptor activation of ERK in rat cortical cultures was partially inhibited by the highly selective NR2B antagonist Ro25-6981 (Ro) and the less selective NR2A antagonist NVP-AAM077 (NVP). When Ro and NVP were added together, inhibition appeared additive and equal to that observed with the NMDA open-channel blocker MK-801. Consistent with a selective coupling of extrasynaptic NMDA receptors to the dominant ERK shutoff pathway, pre-block of synaptic NMDA receptors with MK-801 did not alter the inhibitory effect of bath-applied NMDA on ERK activity. Lastly, in contrast to a complete block of synaptic NMDA receptor activation of ERK by extrasynaptic NMDA receptors, activation of extrasynaptic NMDA receptors had no effect upon ERK activation by brain-derived neurotrophic factor. These results suggest that the synaptic NMDA receptor ERK activation pathway is coupled to both NR2A and NR2B containing receptors, and that the extrasynaptic NMDA receptor ERK inhibitory pathway is not a non-selective global ERK shutoff.

  16. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  17. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

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    John Paul Christianson

    2014-10-01

    Full Text Available Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed behavioral immunization. Recent data implicate neuroplasticity within the ventromedial prefrontal cortex (mPFC as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and one week later to uncontrollable tailshocks, followed 24h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDAR and the extracellular signal-regulated kinase (ERK cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL or infralimbic (IL mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent plasticity within the PL region is required for behavioral immunization, a learned form of stressor resistance.

  18. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

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    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  19. Protein kinase C δ (PKCδ)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition-mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)-induced endotoxemia.

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    Noh, Kyung Tae; Son, Kwang Hee; Jung, In Duk; Kang, Hyun Kyu; Hwang, Sun Ae; Lee, Won Suk; You, Ji Chang; Park, Yeong-Min

    2012-04-20

    Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.

  20. Involvement of extracellular signal regulated kinases in traumatic brain injury-induced depression in rodents.

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    Kuo, Jinn-Rung; Cheng, Yi-Hsuan; Chen, Yi-Shion; Chio, Chung-Ching; Gean, Po-Wu

    2013-07-15

    Traumatic brain injury (TBI) is the most common cause of death and acquired disability among children and young adults in the developed countries. In clinical studies, the incidence of depression is high after TBI, and the mechanisms behind TBI-induced depression remain unclear. In the present study, we subjected rats to a moderate fluid percussion into the closed cranial cavity to induce TBI. After 3 days of recovery, injured rats were given a forced swim test (FST) and novelty-suppressed feeding tests. We found that TBI rats exhibited increased duration of immobility and longer latency to begin chewing food in a new environment compared with sham-operated rats. Western blot analysis showed that TBI led to a decrease in the phosphorylated levels of extracellular signal regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK). Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), significantly reduced the duration of immobility when administered once per day for 14 days. Consistent with behavioral tests, fluoxetine treatment reversed TBI-induced decrease in p-ERK1/2 and p-p38 MAPK levels. Pre-treatment with a selective tryptophan hydroxylase inhibitor para-chlorophenylalanine (PCPA) blocked the antidepressant effect of fluoxetine. PCPA also prevented the effect of fluoxetine on ERK1/2 phosphorylation without affecting p38 MAPK phosphorylation. Pre-treatment with ERK inhibitor SL327 but not p38 MAPK inhibitor SB203580 prevented the antidepressant effect of fluoxetine. These results suggest that ERK1/2 plays a critical role in TBI-induced depression.

  1. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  2. Extracellular Signal-Regulated Kinase Is a Direct Target of the Anti-Inflammatory Compound Amentoflavone Derived from Torreya nucifera

    Directory of Open Access Journals (Sweden)

    Jueun Oh

    2013-01-01

    Full Text Available Amentoflavone is a biflavonoid compound with antioxidant, anticancer, antibacterial, antiviral, anti-inflammatory, and UV-blocking activities that can be isolated from Torreya nucifera, Biophytum sensitivum, and Selaginella tamariscina. In this study, the molecular mechanism underlying amentoflavone’s anti-inflammatory activity was investigated. Amentoflavone dose dependently suppressed the production of nitric oxide (NO and prostaglandin E2 (PGE2 in RAW264.7 cells stimulated with the TLR4 ligand lipopolysaccharide (LPS; derived from Gram-negative bacteria. Amentoflavone suppressed the nuclear translocation of c-Fos, a subunit of activator protein (AP-1, at 60 min after LPS stimulation and inhibited the activity of purified and immunoprecipitated extracellular signal-regulated kinase (ERK, which mediates c-Fos translocation. In agreement with these results, amentoflavone also suppressed the formation of a molecular complex including ERK and c-Fos. Therefore, our data strongly suggest that amentoflavone’s immunopharmacological activities are due to its direct effect on ERK.

  3. Fucoidan from Fucus vesiculosus suppresses hepatitis B virus replication by enhancing extracellular signal-regulated Kinase activation.

    Science.gov (United States)

    Li, Huifang; Li, Junru; Tang, Yuan; Lin, Lin; Xie, Zhanglian; Zhou, Jia; Zhang, Liyun; Zhang, Xiaoyong; Zhao, Xiaoshan; Chen, Zhengliang; Zuo, Daming

    2017-09-16

    Hepatitis B virus (HBV) infection is a serious public health problem leading to cirrhosis and hepatocellular carcinoma. As the clinical utility of current therapies is limited, the development of new therapeutic approaches for the prevention and treatment of HBV infection is imperative. Fucoidan is a natural sulfated polysaccharide that extracted from different species of brown seaweed, which was reported to exhibit various bioactivities. However, it remains unclear whether fucoidan influences HBV replication or not. The HBV-infected mouse model was established by hydrodynamic injection of HBV replicative plasmid, and the mice were treated with saline or fucoidan respectively. Besides, we also tested the inhibitory effect of fucoidan against HBV infection in HBV-transfected cell lines. The result showed that fucoidan from Fucus vesiculosus decreased serum HBV DNA, HBsAg and HBeAg levels and hepatic HBcAg expression in HBV-infected mice. Moreover, fucoidan treatment also suppressed intracellular HBcAg expression and the secretion of the HBV DNA as well as HBsAg and HBeAg in HBV-expressing cells. Furthermore, we proved that the inhibitory activity by fucoidan was due to the activation of the extracellular signal-regulated kinase (ERK) pathway and the subsequent production of type I interferon. Using specific inhibitor of ERK pathway abrogated the fucoidan-mediated inhibition of HBV replication. This study highlights that fucoidan might be served as an alternative therapeutic approach for the treatment of HBV infection.

  4. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  5. Role of reactive oxygen species in extracellular signal-regulated protein kinase phosphorylation and 6-hydroxydopamine cytotoxicity

    Indian Academy of Sciences (India)

    Scott M Kulich; Charleen T Chu

    2003-02-01

    A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes to toxicity elicited by 6-hydroxydopamine (6-OHDA) in the B65 neuronal cell line. To determine whether reactive oxygen species (ROS) play a role in mediating ERK activation and 6-OHDA toxicity, we examined the effects of catalase, superoxide dismutase (SOD1), and metalloporphyrin antioxidants (‘SOD mimetics’) on 6-OHDA-treated cells. We found that catalase and metalloporphyrin antioxidants not only conferred protection against 6-OHDA but also inhibited development of sustained ERK phosphorylation in both differentiated and undifferentiated B65 cells. However, exogenously added SOD1 and heat-inactivated catalase had no effect on either toxicity or sustained ERK phosphorylation. This correlation between antioxidant protection and inhibition of 6-OHDA-induced sustained ERK phosphorylation suggests that redox regulation of ERK signalling cascades may contribute to neuronal toxicity.

  6. A novel role for extracellular signal-regulated kinase in maintaining long-term memory-relevant excitability changes.

    Science.gov (United States)

    Cohen-Matsliah, Sivan Ida; Brosh, Inbar; Rosenblum, Kobi; Barkai, Edi

    2007-11-14

    Pyramidal neurons in the piriform cortex from olfactory-discrimination-trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the postburst afterhyperpolarization (AHP), which is generated by repetitive spike firing. AHP reduction is attributable to decreased conductance of a calcium-dependent potassium current, the sI(AHP). We have previously shown that such learning-induced AHP reduction is maintained by PKC activation. However, the molecular machinery underlying such long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the extracellular signal-regulated kinase I/II (ERKI/II) pathway, which is known to be crucial in learning, memory, and synaptic plasticity processes, is instrumental for the long-term maintenance of learning-induced AHP reduction. PD98059 or UO126, which selectively block MEK, the upstream kinase of ERK, increased the AHP in neurons from trained rats but not in neurons from naive and pseudo-trained rats. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls were abolished. This effect was not mediated by modulation of basic membrane properties. In accordance with its effect on neuronal excitability, the level of activated ERK in the membranal fraction was significantly higher in piriform cortex samples taken from trained rats. In addition, the PKC activator OAG (1-oleoyl-20acety-sn-glycerol), which was shown to reduce the AHP in neurons from control rats, had no effect on these neurons in the presence of PD98059. Our data show that ERK has a key role in maintaining long-lasting learning-induced enhancement of neuronal excitability.

  7. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S;

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen...

  8. α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    Full Text Available G protein-coupled receptors (GPCRs activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A-adrenergic receptor (α(1A-AR-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2. Agonist-mediated endocytic traffic of α(1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A. α(1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A-AR. α(1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent. Activation of protein kinase C (PKC and C-Raf by α(1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor and Ro 31-8220 (a PKC inhibitor inhibited α(1B-AR- but not α(1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A-AR-induced ERK1/2 activation, which is independent of G(q/PLC/PKC signaling.

  9. Regulation of extracellular signal-regulated kinase 1/2 inlfuences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Yaning Zhao; Jianmin Li; Qiqun Tang; Pan Zhang; Liwei Jing; Changxiang Chen; Shuxing Li

    2014-01-01

    Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor (U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and Ku70 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These ifndings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac-celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/reperfusion.

  10. 脑缺血时NMDA受体通过Src激酶和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ调控ERKs激活%N-methyl-D-aspartate receptors mediate diphosphorylation of extracellular signal-regulated kinases through Src family tyrosine kinases and Ca2+/calmodulin-dependent protein kinase Ⅱ in rat hippocampus after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    吴辉文; 李洪福; 郭军

    2007-01-01

    目的 ERKs是钙依赖性激活蛋白,本研究旨在探讨钙依赖性蛋白激酶是否参与了脑缺血后ERK级联的调控.方法 采用四动脉结扎诱导大鼠前脑缺血,用免疫印迹的方法观察几个钙依赖性蛋白激酶含量及活性的变化.结果 致死性脑缺血以NMDA受体依赖的方式激活ERKs,并差异性上调Src和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的活性.Src激酶和CaMKⅡ的抑制剂PP2和KN62能显著的阻止缺血诱导的ERKs激活.然而,缺血诱导的Src过度激活也伴随着ERKs的活性抑制.结论 致死性脑缺血刺激NMDA受体通过Src激酶和CaMKⅡ介导ERKs活性上调,但是脑缺血诱导的Src过度激活可能也参与了ERKs信号通路的负性调控.%Objective: Extracellular signal-regulated kinases (ERKs) can be activated by calcium signals. In this study, we investigated whether calcium-dependent kinases were involved in ERKs cascade activation after global cerebral ischemia.Methods Cerebral ischemia was induced by four-vessel occlusion, and the calcium-dependent proteins were detected by immunoblot. Results Lethal-simulated ischemia significantly resulted in ERKs activation in N-methyl-D-aspartate (NMDA)receptor-dependent manner, accompanying with differential upregulation of Src kinase and Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) activities. With the inhibition of Src family tyrosine kinases or CaMKⅡ by administration of PP2or KN62, the phosphorylation of ERKs was impaired dramatically during post-ischemia recovery. However, ischemic challenge also repressed ERKs activity when Src kinase was excessively activated. Conclusions Src family tyrosine kinases and CaMKⅡ might be involved in the activation of ERKs mediated by NMDA receptor in response to acute ischemic stimuli in vivo, but the intense activation of Src kinase resulted from ischemia may play a reverse role in the ERKs cascade.

  11. Differential extracellular signal-regulated kinases 1 and 2 activation by the angiotensin type 1 receptor supports distinct phenotypes of cardiac myocytes

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael;

    2007-01-01

    The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains...... that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus......, the beta-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling...

  12. Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

    Science.gov (United States)

    Dinicola, Simona; Mariggiò, Maria Addolorata; Morabito, Caterina; Guarnieri, Simone; Cucina, Alessandra; Pasqualato, Alessia; D'Anselmi, Fabrizio; Proietti, Sara; Coluccia, Pierpaolo; Bizzarri, Mariano

    2013-09-14

    Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

  13. The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz-Cuesta, Elena; Olesen, Christina W;

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

  14. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner. CONCLUSIONS: This work characterizes a new type of scaffolding complex, which we term a "shuffle complex", between the disordered hNHE1-tail...

  15. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder.

    Science.gov (United States)

    Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi

    2013-03-25

    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  16. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signaltransduction pathway in depressive disorder

    Institute of Scientific and Technical Information of China (English)

    Hongyan Wang; Yingquan Zhang; Mingqi Qiao

    2013-01-01

    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  17. N-glycosylation at Asn residues 554 and 566 of E-cadherin affects cell cycle progression through extracellular signal-regulated protein kinase signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Hongbo Zhao; Xiliang Zha; Lidong Sun; Liying Wang; Zhibin Xu; Feng Zhou; Jianmin Su; Jiawei Jin; Yong Yang; Yali Hu

    2008-01-01

    E-cadherin, which has a widely acknowledged role in mediating calcium-dependent cell-cell adhesion between epithelial cells, also functions as a tumor suppressor. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633.We investigated the role of E-cadherin N-glycosylation in cell cycle progression by site-directed mutagenesis. We showed previously that all four potential N-glycosylation sites of E-cadherin were N-glycosylated in human breast carcinoma MDA-MB-435 cells. Removal of N-glycan at Asn633 dramatically affected E-cadherin stability. In this study we showed that E-cadherin mutant missing N-glycans at Asn554, Asn566 and Asn618 failed to induce cell cycle arrest in G1 phase and to suppress cell proliferation in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn554 and Asn566, but not at Asn618, seemed to be indispensable for E-cadherin-mediated suppression of cell cycle progression.Removal of N-glycans at either Asn554 or Asn566 of E-cadherin was accompanied with the activation of the extracellular signal-regulated protein kinase signaling pathway. After treatment with PD98059, an inhibitor of the extraceilular signal-regulated protein kinase signaling pathway, wild-type E-cadherin transfected MDA-MB-435 and E-cadherin N-glycosylation-deficient mutant transfected MDA-MB-435 cells had equivalent numbers of cells in G1 phase. These findings implied that N-glycosylation might be crucial for E-cadherin-mediated suppression of cell cycle progression.

  18. Activation of Extracellular Signal-Regulated Kinases (ERK 1/2) in the Locus Coeruleus Contributes to Pain-Related Anxiety in Arthritic Male Rats.

    Science.gov (United States)

    Borges, Gisela; Miguelez, Cristina; Neto, Fani; Mico, Juan Antonio; Ugedo, Luisa; Berrocoso, Esther

    2017-06-01

    There is increasing evidence suggesting that the Locus Coeruleus plays a role in pain-related anxiety. Indeed, we previously found that prolonged arthritis produces anxiety-like behavior in rats, along with enhanced expression of phosphorylated extracellular signal-regulated kinase 1/2 (a marker of plasticity) in the Locus Coeruleus. However, it is unknown how this effect correlates with the electrophysiological activity of Locus Coeruleus neurons or pain-related anxiety. Using the complete Freund's adjuvant model of monoarthritis in male Sprague-Dawley rats, we studied the behavioral attributes of pain and anxiety as well as Locus Coeruleus electrophysiology in vivo 1 (MA1W) and 4 weeks (MA4W) after disease induction. The manifestation of anxiety in MA4W was accompanied by dampened tonic Locus Coeruleus activity, which was coupled to an exacerbated evoked Locus Coeruleus response to noxious stimulation of the inflamed and healthy paw. When a mitogen-activating extracellular kinase inhibitor was administered to the contralateral Locus Coeruleus of MA4W, the phosphorylated extracellular signal-regulated kinase 1/2 levels in the Locus Coeruleus were restored and the exaggerated evoked response was blocked, reversing the anxiogenic-like behavior while pain hypersensitivity remained unaltered. As phosphorylated extracellular signal-regulated kinase 1/2 blockade in the Locus Coeruleus relieved anxiety and counteracted altered LC function, we propose that phosphorylated extracellular signal-regulated kinase 1/2 activation in the Locus Coeruleus plays a crucial role in pain-related anxiety.

  19. Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

    Science.gov (United States)

    Lai, Xiangru; Ye, Lingyan; Liao, Yuan; Jin, Lili; Ma, Qiang; Lu, Bing; Sun, Yi; Shi, Ying; Zhou, Naiming

    2016-04-01

    The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here

  20. Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Zhengyu [Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040 (China); Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437 (China); Yang, Qi; Cui, Mei; Liu, Yanping [Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040 (China); Wang, Tao; Zhao, Hong [Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437 (China); Dong, Qiang, E-mail: qiang_dong163@163.com [Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040 (China)

    2014-03-28

    Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depleted of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.

  1. Dopamine D1 Receptors Regulate Protein Synthesis-Dependent Long-Term Recognition Memory via Extracellular Signal-Regulated Kinase 1/2 in the Prefrontal Cortex

    Science.gov (United States)

    Nagai, Taku; Takuma, Kazuhiro; Kamei, Hiroyuki; Ito, Yukio; Nakamichi, Noritaka; Ibi, Daisuke; Nakanishi, Yutaka; Murai, Masaaki; Mizoguchi, Hiroyuki; Nabeshima, Toshitaka; Yamada, Kiyofumi

    2007-01-01

    Several lines of evidence suggest that extracellular signal-regulated kinase1/2 (ERK1/2) and dopaminergic system is involved in learning and memory. However, it remains to be determined if the dopaminergic system and ERK1/2 pathway contribute to cognitive function in the prefrontal cortex (PFC). The amount of phosphorylated ERK1/2 was increased in…

  2. 反义细胞外信号调节激酶-2基因治疗移植物动脉血管病内膜病变%The effect of adenovirus-mediated anti-extracellular signal regulated kinase 2 gene therapy on intimal change in transplant arteriosclerosis

    Institute of Scientific and Technical Information of China (English)

    赵波; 宫念樵

    2011-01-01

    目的 观察移植物动脉血管病(TA)的内膜病变机制和反义细胞外信号调节激酶2基因腺病毒载体(Adanti-ERK2)基因治疗的效果.方法 建立Brown-Norway(BN)-Lewis移植物动脉血管病模型,分为同系组、Control组、LacZ组和Adanti-ERK2组(给予5×109 pfu Adanti-ERK2基因治疗),每组各6例.术后60 d检测各组内膜病变和血管腔内膜/(内膜+中膜)比,α-肌动蛋白(α-actin)和血小板源性生长因子-BB(PDGF-BB)染色检测移植动脉平滑肌细胞(VSMCs)增殖和分泌功能,评估移植动脉新生毛细血管情况并检测移植动脉中环氧化酶-2(COX-2)的表达.结果 术后60 d同系组内膜无异常,Control组和LacZ组典型内膜增殖改变,Adanti-ERK2组内膜病变较轻;内膜/(内膜+中膜)比各组分别为7.6%、81.4%、85.9%、15.9%;α-actin阳性细胞(内膜平滑肌细胞)每视野计数各组分别为0、71.3±9.2、76.4±11.3、34.8±5.3;PDGF-BB阳性细胞每视野计数各组分别为0.9±0.5、28.4±3.4、29.1±3.2、8.6±1.7;移植动脉中膜和内膜新生毛细血管检测各组分别无、丰富、丰富、少量;COX-2新生血管阳性细胞计数各组分别为0、36.3±8.3、40.9±9.2、10.4±3.9.Adanti-ERK2组与其他组别间比较,差异有统计学意义(P<0.05).结论 内膜增生,血管腔缩窄,PDGF-BB诱导内膜平滑肌细胞募集分化并激发血管新生是TA重要病理生理环节,AdantiERK2基因治疗可有效干预各发病环节,达到治疗效果.%Objective To explore the mechanisms of intimal injury underlying transplant arteriosclerosis (TA) and to clarify the treatment effect of adenovirus-mediated anti-extracellular signal regulated kinase 2 (Adanti-ERK2) gene therapy on TA. Methods The Brown-Norway (BN)-Lewis TA model was employed. According to different gene therapy, the recipients were divided into isograft group, control group, LacZ group, which were used as control, and Adanti-ERK2 group (5 × 109 pfu Adanti-ERK2 was transferred

  3. Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

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    Margarida Ressurreição

    2014-06-01

    Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

  4. Traditional Chinese medicine suppresses left ventricular hypertrophy by targeting extracellular signal-regulated kinases signaling pathway in spontaneously hypertensive rats

    Science.gov (United States)

    Xiong, Xingjiang; Yang, Xiaochen; Duan, Lian; Liu, Wei; Zhang, Yun; Liu, Yongmei; Wang, Pengqian; Li, Shengjie; Li, Xiaoke

    2017-01-01

    Chinese herbal medicine Bu-Shen-Jiang-Ya decoction (BSJYD) is reported to be beneficial for hypertension. Over expression of extracellular signal regulated kinases (ERK) pathway plays an important role in left ventricular hypertrophy (LVH). This study aimed to observe effects of BSJYD on LVH in spontaneously hypertensive rats (SHRs) and explore its possible mechanism on regulation of ERK pathway. Sixty 12-week-old SHRs were randomly allocated into 5 groups: BSJYD high dose group, middle dose group, low dose group, captopril group, and control group. Besides, a control group of Wistar-Kyoto rats was established. All rats were treated for 8 weeks. Systolic blood pressure (SBP), heart rate (HR), pathology, and left ventricular mass index (LVMI) were measured. Western blotting and Real-time PCR were used to assess the expressions of BDNF, Ras, ERK1/2, and c-fox levels. SBP and HR were significantly decreased compared with the control group and LVMI was markedly improved by BSJYD treatment in a dose-dependent manner. BSJYD inhibited the expression of BDNF, Ras, ERK1/2, and c-fox mRNA in LVH. In conclusion, BSJYD suppressed hypertension-induced cardiac hypertrophy by inhibiting the expression of ERK pathway. These changes in gene expression may be a possible mechanism by which BSJYD provides myocardial protection from hypertension. PMID:28225023

  5. Pleiotrophin promotes microglia proliferation and secretion of neurotrophic factors by activating extracellular signal-regulated kinase 1/2 pathway.

    Science.gov (United States)

    Miao, Jiayin; Ding, Minghui; Zhang, Aiwu; Xiao, Zijian; Qi, Weiwei; Luo, Ning; Di, Wei; Tao, Yuqian; Fang, Yannan

    2012-12-01

    Pleiotrophin (PTN) is an effective neuroprotective factor and its expression is strikingly increased in microglia after ischemia/reperfusion injury. However, whether PTN could provide neurotrophic support to neurons by regulating microglia function is not clear. In this study, we demonstrated that the expression of PTN was induced in microglia after oxygen-glucose deprivation/reperfusion. PTN promoted the proliferation of microglia by enhancing the G1 to S phase transition. PTN also stimulated the secretion of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) in microglia, but did not upregulate the expression of proinflammatory factors such as TNF-α, IL-1β and iNOS. Mechanistically, we found that PTN increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in microglia in both concentration-dependent and time-dependent manners. In addition, ERK1/2 inhibitor U0126 abolished the proliferation and G1 to S phase transition of microglia stimulated by PTN, and inhibited the production of BDNF, CNTF and NGF induced by PTN. In conclusion, our results demonstrated that PTN-ERK1/2 pathway plays important role in regulating microglia growth and secretion of neurotrophic factors. These findings provide new insight into the neuroprotective role of PTN and suggest that PTN is a new target for therapeutic intervention of stroke.

  6. Epidermal Growth Factor Stimulates Extracellular-Signal Regulated Kinase Phosphorylation of a Novel Site on Cytoplasmic Dynein Intermediate Chain 2

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    Andrew D. Catling

    2013-02-01

    Full Text Available Extracellular-signal regulated kinase (ERK signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2 as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

  7. Inhibition of Extracellular Signal-Regulated Kinases Ameliorates Hypertension-Induced Renal Vascular Remodeling in Rat Models

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    Li Jing

    2011-11-01

    Full Text Available The aim of this study is to investigate the effect of the extracellular signal-regulated kinases 1/2 (ERK1/2 inhibitor, PD98059, on high blood pressure and related vascular changes. Blood pressure was recorded, thicknesses of renal small artery walls were measured and ERK1/2 immunoreactivity and erk2 mRNA in renal vascular smooth muscle cells (VSMCs and endothelial cells were detected by immunohistochemistry and in situ hybridization in normotensive wistar kyoto (WKY rats, spontaneously hypertensive rats (SHR and PD98059-treated SHR. Compared with normo-tensive WKY rats, SHR developed hypertension at 8 weeks of age, thickened renal small artery wall and asymmetric arrangement of VSMCs at 16 and 24 weeks of age. Phospho-ERK1/2 immunoreactivity and erk2 mRNA expression levels were increased in VSMCs and endothelial cells of the renal small arteries in the SHR. Treating SHR with PD98059 reduced the spontaneous hypertension-induced vascular wall thickening. This effect was associated with suppressions of erk2 mRNA expression and ERK1/2 phosphorylation in VSMCs and endothelial cells of the renal small arteries. It is concluded that inhibition of ERK1/2 ameliorates hypertension induced vascular remodeling in renal small arteries.

  8. Extracellular signal-regulated kinase activation in spinal astrocytes and microglia contributes to cancer-induced bone pain in rats.

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    Wang, X-W; Li, T-T; Zhao, J; Mao-Ying, Q-L; Zhang, H; Hu, S; Li, Q; Mi, W-L; Wu, G-C; Zhang, Y-Q; Wang, Y-Q

    2012-08-16

    Cancer pain, especially cancer-induced bone pain, affects the quality of life of cancer patients, and current treatments for this pain are limited. The present study demonstrates that spinal extracellular signal-regulated kinase (ERK) activation in glial cells plays a crucial role in cancer-induced bone pain. From day 4 to day 21 after the intra-tibia inoculation with Walker 256 mammary gland carcinoma cells, significant mechanical allodynia was observed as indicated by the decrease of mechanical withdrawal thresholds in the von Frey hair test. Intra-tibia inoculation with carcinoma cells induced a vast and persistent (>21 D) activation of ERK in the bilateral L2-L3 and L4-L5 spinal dorsal horn. The increased pERK1/2-immunoreactivity was observed in both Iba-1-expressing microglia and GFAP-expressing astrocytes but not in NeuN-expressing neurons. A single intrathecal injection of the selective MEK (ERK kinase) inhibitors PD98059 (10 μg) on day 12 and U0126 (1.25 and 3 μg) on day 14, attenuated the bilateral mechanical allodynia in the von Frey hair test. Altogether, our results suggest that ERK activation in spinal microglia and astrocytes is correlated with the onset of allodynia and is important for allodynia maintenance in the cancer pain model. This study indicated that inhibition of the ERK pathway may provide a new therapy for cancer-induced bone pain.

  9. Extracellular signal-regulated kinases (ERKs) pathway and reactive oxygen species regulate tyrosine phosphorylation in capacitating boar spermatozoa.

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    Awda, Basim J; Buhr, Mary M

    2010-11-01

    The extracellular signal-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) pathway is identified for the first time in boar sperm and is associated with capacitation and tyrosine phosphorylation (tyr-P). Reactive oxygen species (ROS) modulate this signal transduction. Western immunoblotting detected the ERK pathway components RAF1, MEK1/2, and ERK1/2 in extracts from fresh boar spermatozoa and determined that their phosphoprotein profiles differed in a capacitation-dependent fashion. Capacitation was accompanied by appearance of two new ERKs (158 and 161 kDa) and disappearance of others. Capacitation was verified with increased tyr-P, which was inhibited by a 30-min pre-exposure of fresh boar sperm to a xanthine/xanthine oxidase ROS-generating system prior to the capacitating incubation; ROS pre-exposure also affected the phosphorylation of RAF1, MEK1/2, and ERK1/2. Preincubating sperm with inhibitors of the ERK components with or without the ROS generator affected subsequent capacitation. Inhibiting ERK1/2 inhibited tyr-P of capacitated boar spermatozoa proteins of 172, 97, and 66 kDa (P ≤ 0.04); with ROS, this inhibition increased (P influence through crosstalk with different pathways. ROS affect RAF1, MEK1/2, and ERK1/2 and could influence the sequential events of boar sperm capacitation.

  10. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

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    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  11. [Expression of extracellular signal-regulated protein kinases in the subcutaneous fascia of rats and their changes after acupuncture].

    Science.gov (United States)

    Jiang, Xue-mei; Yang, Chun; Yuan, Lin; Diao, Jian-xin; Zhang, Xue-quan; Huang, Yong; Dai, Jing-xing; Qiu, Xiao-zhong; Yu, Lei

    2009-04-01

    To observe the effect of acupuncture on the expression of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in the subcutaneous fascia of SD rats. Eighteen SD rats were randomly divided into 6 groups (n=3) including 5 acupuncture groups and a control group. The rats in the 5 acupuncture groups received electro-acupuncture therapy in the regions of the inguinal groove, and at 0, 1, 6, 12, and 36 h after the last therapy, the superfacial fascia surrounding the acupuncture point (about 1.5 cm in diameter) were collected. The fascia tissues at the corresponding sites and at the acupoint Zusanli (ST36) were obtained from the control rats. The expression of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the tissues were detected by Western blotting. ERK1/2 and p-ERK1/2 expressions were detected in the tissues harvested from both the acupoint and the non-acupoint in the control rats with similar expression intensities. In the rats of each acupuncture group, ERK1/2 expression was significantly increased on the acupuncture side in comparison with the control side. The normal loose connective tissue may participate in tissue proliferation and differentiation possibly via phosphorylation of ERK. Acupuncture can promote the signal transduction pathway of ERK, which can be a possible mechanism for the effect of acupuncture in modulating the physiopathological conditions.

  12. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

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    Friedman, Adam A; Tucker, George; Singh, Rohit; Yan, Dong; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L; Asara, John M; Berger, Bonnie; Perrimon, Norbert

    2011-10-25

    Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

  13. Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro

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    Juan Peng

    2015-12-01

    Full Text Available AIM: To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK signaling pathway in vitro. METHODS: The expression levels of phosphorylated ERK (P-ERK, keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis. RESULTS: The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression. CONCLUSION: We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.

  14. Expression of Extracellular Signal-regulated Kinase and Angiotensin-converting Enzyme in Human Atria during Atrial Fibrillation

    Institute of Scientific and Technical Information of China (English)

    戴友平; 王祥; 曹林生; 杨杪; 邬堂春

    2004-01-01

    Summary: In order to investigate the changes in the expression of extracellular signal-regulated kinase (ERK1/ERK2) and angiotensin-converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK-activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2-mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P<0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three-fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE-dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.

  15. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Energy Technology Data Exchange (ETDEWEB)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  16. Role of extracellular signal-regulated kinase in regulating expression of interleukin 13 in lymphocytes from an asthmatic rat model

    Institute of Scientific and Technical Information of China (English)

    LI Yuan-yuan; LIU Xian-sheng; LIU Chang; XU Yong-jian; XIONG Wei-xing

    2010-01-01

    Background The extracellular signal-regulated kinase (ERK) is widely expressed in mammal cells and involved in airway proliferation and remodeling in asthma. In this study, we intend to explore the role of ERK in the expression of the Th2 cytokine, interleukin 13 (IL-13) in lymphocytes in asthma.Methods Forty Sprague-Dawley rats were randomly divided into two groups: normal control and asthmatic groups. Peripheral blood lymphocytes were isolated and purified from the blood of each rat and divided into five groups: control, asthmatic lymphocytes, asthmatic cells stimulated with ERK activator epidermal growth factor (EGF), or with ERK inhibitor PD98059, or with EGF and PD98059 together. The expression of phosphorylated-ERK (p-ERK) was observed by immunocvtochemical staining, the expression of ERK mRNA was determined by reverse transcriptase-PCR, IL-13 protein in supernatants was measured by ELISA.Results (1) The ERK mRNA level and the percentage of cells with p-ERK in lymphocytes from asthmatic rats were significantly higher than those in normal controls, and were significantly increased by EGF administration. This effect of EGF was significantly inhibited by PD98059 pretreatment. (2) IL-13 protein in supematants of asthmatic lymphocytes was higher than that produced by normal control lymphocytes, and was significantly increased by EGF treatment. This EGF effect was partly blocked by PD98059 pretreatment. (3) There was a significant positive correlation between the percentage of cells with p-ERK in peripheral blood lymphocytes and IL-13 protein in supematants of lymphocytes from asthmatic rats.Conclusions In asthma the ERK expression and activation levels were increased, as was the protein level of IL-13. The ERK signaling pathway may be involved in the increased expression of the Th2 cytokine IL-13 in asthma.

  17. Cue-elicited reward-seeking requires extracellular signal-regulated kinase activation in the nucleus accumbens.

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    Shiflett, Michael W; Martini, Ross P; Mauna, Jocelyn C; Foster, Rebecca L; Peet, Eloise; Thiels, Edda

    2008-02-01

    The motivation to seek out rewards can come under the control of stimuli associated with reward delivery. The ability of cues to motivate reward-seeking behavior depends on the nucleus accumbens (NAcc). The molecular mechanisms in the NAcc that underlie the ability of a cue to motivate reward-seeking are not well understood. We examined whether extracellular signal-regulated kinase (ERK), an important intracellular signaling pathway in learning and memory, has a role in these motivational processes. We first examined p42 ERK (ERK2) activation in the NAcc after rats were trained to associate an auditory stimulus with food delivery and found that, as a consequence of training, presentation of the auditory cue itself was sufficient to increase ERK2 activation in the NAcc. To examine whether inhibition of ERK in the NAcc prevents cue-induced reward-seeking, we infused an inhibitor of ERK, U0126, into the NAcc before assessing rats' instrumental responding in the presence versus absence of the conditioned cue. We found that, whereas vehicle-infused rats showed increased instrumental responding during cue presentation, rats infused with U0126 showed a profound impairment in cue-induced instrumental responding. In contrast, intra-NAcc U0126 infusion had no effect on rats' food-reinforced instrumental responding or their ability to execute conditioned approach behavior. Our results demonstrate learning-related changes in ERK signaling in the NAcc, and that disruption of ERK activation in this structure interferes with the incentive-motivational effects of conditioned stimuli. The molecular mechanisms described here may have implications for cue-elicited drug craving after repeated exposure to drugs of abuse.

  18. Short waves-induced enhancement of proliferation of human chondrocytes: involvement of extracellular signal-regulated map-kinase (erk).

    Science.gov (United States)

    Wang, Jue-Long; Chan, Rai-Chi; Cheng, He-Hsiung; Huang, Chun-Jen; Lu, Yih-Chau; Chen, I-Shu; Liu, Shiuh-Inn; Hsu, Shu-Shong; Chang, Hong-Tai; Huang, Jong-Khing; Chen, Jin-Shyr; Ho, Chin-Man; Jan, Chung-Ren

    2007-07-01

    1. Short-wave diathermy (SWD) is a form of radiofrequency radiation that is used therapeutically by physiotherapists. The cellular mechanisms of SWD are unclear. The present study was performed to explore the effect of different conditions of short-wave exposure on the proliferation of cultured human chondrocytes. 2. Cells exposed to short waves once per day for seven consecutive days exhibited a significant increase in proliferation by 42% compared with the control cells. In cells that were treated with short waves twice per day for seven consecutive days, or only once on Day 1 and then examined for proliferation on Day 7, cell proliferation was greater than the control cells by 40% and 30%, respectively. 3. Given the importance of mitogen-activated protein kinases (MAPK) in the proliferation of different cell types, efforts were extended to explore the role of three major types of MAPK; that is, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal protein kinase (JNK) and p38. 4. It was found that the level of phosphorylated ERK (phospho-ERK 1 and ERK 2) increased significantly within 5-120 min following consecutive exposure to short waves for 7 days. Exposure to short waves failed to alter the intensity of phosphorylated JNK and p38 within 0-240 min. 5. Cells were exposed to short waves once for seven consecutive days in the presence of 0, 10 micromol/L, 20 micromol/L or 50 micromol/L PD98059 (an ERK inhibitor). PD98059 totally inhibited short waves-induced enhancement of proliferation without altering normal control viability. In the presence of short waves and PD98059, the cell viability was lower than the normal control. Together, the data suggest that short waves could increase proliferation in human chondrocytes through activation of the ERK pathway, which is also involved in maintaining normal cell proliferation under physiological conditions.

  19. Effects of chronic sleep deprivation on the extracellular signal-regulated kinase pathway in the temporomandibular joint of rats.

    Directory of Open Access Journals (Sweden)

    Chuan Ma

    Full Text Available OBJECTIVES: To examine the possible involvement and regulatory mechanisms of extracellular signal-regulated kinase (ERK pathway in the temporomandibular joint (TMJ of rats subjected to chronic sleep deprivation (CSD. METHODS: Rats were subjected to CSD using the modified multiple platform method (MMPM. The serum levels of corticosterone (CORT and adrenocorticotropic hormone (ACTH were tested and histomorphology and ultrastructure of the TMJ were observed. The ERK and phospho-ERK (p-ERK expression levels were detected by Western blot analysis, and the MMP-1, MMP-3, and MMP-13 expression levels were detected by real-time quantitative polymerase chain reaction (PCR and Western blotting. RESULTS: The elevated serum CORT and ACTH levels confirmed that the rats were under CSD stress. Hematoxylin and eosin (HE staining and scanning electron microscopy (SEM showed pathological alterations in the TMJ following CSD; furthermore, the p-ERK was activated and the mRNA and protein expression levels of MMP-1, MMP-3, and MMP-13 were upregulated after CSD. In the rats administered with the selective ERK inhibitor U0126, decreased tissue destruction was observed. Phospho-ERK activation was visibly blocked and the MMP-1, MMP-3, and MMP-13 mRNA and protein levels were lower than the corresponding levels in the CSD without U0126 group. CONCLUSION: These findings indicate that CSD activates the ERK pathway and upregulates the MMP-1, MMP-3, and MMP-13 mRNA and protein levels in the TMJ of rats. Thus, CSD induces ERK pathway activation and causes pathological alterations in the TMJ. ERK may be associated with TMJ destruction by promoting the expression of MMPs.

  20. Involvement of activation of NADPH oxidase and extracellular signal-regulated kinase (ERK) in renal cell injury induced by zinc.

    Science.gov (United States)

    Matsunaga, Yoshiko; Kawai, Yoshiko; Kohda, Yuka; Gemba, Munekazu

    2005-05-01

    Zinc is employed as a supplement; however, zinc-related nephropathy is not generally known. In this study, we investigated zinc-induced renal cell injury using a pig kidney-derived cultured renal epithelial cell line, LLC-PK(1), with proximal kidney tubule-like features, and examined the involvement of free radicals and extracellular signal-regulated kinase (ERK) in the cell injury. The LLC-PK(1) cells showed early uptake of zinc (30 microM), and the release of lactate dehydrogenase (LDH), an index of cell injury, was observed 24 hr after uptake. Three hours after zinc exposure, generation of reactive oxygen species (ROS) was increased. An antioxidant, N, N'-diphenyl-p-phenylenediamine (DPPD), inhibited a zinc-related increase in ROS generation and zinc-induced renal cell injury. An NADPH oxidase inhibitor, diphenyleneiodonium (DPI), inhibited a zinc-related increase in ROS generation and cell injury. We investigated translocation from the cytosol fraction of the p67(phox) subunit, which is involved in the activation of NADPH oxidase, to the membrane fraction, and translocation was induced 3 hr after zinc exposure. We examined the involvement of ERK1/2 in the deterioration of zinc-induced renal cell injury, and the association between ERK1/2 and an increase in ROS generation. Six hours after zinc exposure, the activation (phosphorylation) of ERK1/2 was observed. An antioxidant, DPPD, inhibited the zinc-related activation of ERK1/2. An MAPK/ERK kinase (MEK1/2) inhibitor, U0126, almost completely inhibited zinc-related cell injury (the release of LDH), but did not influence ROS generation. These results suggest that early intracellular uptake of zinc by LLC-PK(1) cells causes the activation of NADPH oxidase, and that ROS generation by the activation of the enzyme leads to the deterioration of renal cell injury via the activation of ERK1/2.

  1. Role of phosphatase PTEN in the activation of extracellular signal-regulated kinases induced by estradiol in endometrial carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    张育军; 魏丽惠; 王建六; 孙铁铮

    2003-01-01

    Objectives To study extracellular signal-regulated kinase (ERK) activation in the endometrial carcinoma cell line Ishikawa with stimulation by 17-β-estradiol, and to elucidate the role of phosphatase and tensin homologue (PTEN) and estrogen receptor (ER) subtype on the activation of ERKs.Methods Western blot was used to examine the expression of PTEN and PTEN (G129E) in Ishikawa cells after stable transfection as well as ERK activation in Ishikawa-EGFP, Ishikawa- PTEN and Ishikawa- PTEN (G129E) stimulated with various doses of 17-β-estradiol for different lengths of time. Western blot was also used for examining the expression of ERα and ERβ in NIH3T3 fibroblasts after transient transfection of pCXN2hERα and pCXN2hERβ. Then, ERK activation was examined after stimulation with 17-β-estradiol. Results 17-β-estradiol activated ERK cascades (mainly ERK2) in Ishikawa cells. The activation of ERK increased gradually as concentration of 17-β-estradiol also increased. The maximal activation of ERK2 took place 5 min after stimulation with 17-β-estradiol. The activation of ERK2 was inhibited markedly by PTEN, but not by PTEN (G129E). 17-β-estradiol activated ERK cascades in NIH3T3 fibroblasts after transient transfection of pCXN2hERα.Conclusions 17-β-estradiol activate ERK cascades in Ishikawa cells by integrating with ERα. Lipid phosphatase PTEN has an inhibitory role on the activation of ERK stimulated by 17-β-estradiol in Ishikawa cells.

  2. An experimental study of extracellular signal-regulated kinase and its inter ventional treatments in hepatic ifbrosis

    Institute of Scientific and Technical Information of China (English)

    Ming-De Jiang; Shu-Mei Zheng; Hui Xu; Wei-Zheng Zeng; Yong Zhang; Hao-Ping Sun; Yun-Xia Wang; Jian-Ping Qin; Xiao-Ling Wu

    2008-01-01

    BACKGROUND:The pathogenesis of hepatic ifbrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a speciifc inhibitor of ERK, on the cell cycle, cell proliferation, secretion of typeⅠcollagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-β1 (TGF-β1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS:Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by lfow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-β1 was examined using the reverse transcriptase-polymerase chain reaction. Type Ⅰcollagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 μmol/L PD98059 signiifcantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of typeⅠ collagen and the expression of cyclin D1, CDK4 and TGF-β1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100μmol/L PD98059, respectively. CONCLUSIONS:The ERK pathway regulates the cell proliferation, secretion of type Ⅰ collagen and the expression of TGF-β1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.

  3. Activation of extracellular signal-regulated kinase (ERK) signaling in the pedunculopontine tegmental (PPT) cells is involved in the maintenance of sleep in rats

    Science.gov (United States)

    Desarnaud, Frank; Macone, Brian W.; Datta, Subimal

    2010-01-01

    Considerable evidence suggests that receptor-mediated excitation and inhibition of brainstem pedunculopontine tegmental (PPT) neurons are critically involved in the regulation of sleep-wake states. However, the molecular mechanisms operating within the PPT controlling sleep-wake states remain relatively unknown. This study was designed to examine sleep-wake state-associated extracellular-signal-regulated kinase 1 and 2 (ERK1/2) transduction changes in the PPT of freely moving rats. The results of this study demonstrate that the levels of ERK1/2 expression, phosphorylation, and activity in the PPT increased with increased amount of time spent in sleep. The sleep-associated increases in ERK1/2 expression, phosphorylation, and activity were not observed in the cortex, or in the immediately adjacent medial pontine reticular formation. The results of regression analyses revealed significant positive relationships between the levels of ERK1/2 expression, phosphorylation, and activity in the PPT and amounts of time spent in slow-wave sleep, rapid eye movement sleep, and total sleep. Additionally, these regression analyses revealed significant negative relationships between the levels of ERK1/2 expression, phosphorylation, and activity in the PPT and amounts of time spent in wakefulness. Collectively, these results, for the first time, suggest that the increased ERK1/2 signaling in the PPT is associated with maintenance of sleep via suppression of wakefulness. PMID:21166678

  4. Mepivacaine-induced contraction involves phosphorylation of extracellular signal-regulated kinase through activation of the lipoxygenase pathway in isolated rat aortic smooth muscle.

    Science.gov (United States)

    Lee, Hyo Min; Ok, Seong-Ho; Sung, Hui-Jin; Eun, So Young; Kim, Hye Jung; Lee, Soo Hee; Kang, Sebin; Shin, Il-Woo; Lee, Heon Keun; Chung, Young-Kyun; Choi, Mun-Jeoung; Bae, Sung Il; Sohn, Ju-Tae

    2013-04-01

    Mepivacaine is an aminoamide local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. This study investigated the arachidonic acid metabolic pathways involved in mepivacaine-induced contraction, and elucidated the associated cellular mechanism with a particular focus on extracellular signal-regulated kinase (ERK) in endothelium-denuded rat aorta. Isolated rat thoracic aortic rings were suspended for isometric tension recording. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, indomethacin, NS-398, SC-560, fluconazole, PD 98059, and verapamil. Mepivacaine-induced ERK phosphorylation, 5-lipoxygenase (5-LOX) expression, and cyclooxygenase (COX)-2 expression in rat aortic smooth muscle cells were detected by Western blot analysis in the presence or absence of inhibitors. Mepivacaine produced tonic contraction in isolated endothelium-denuded rat aorta. Quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, NS-398, PD 98059, and verapamil attenuated mepivacaine-induced contraction in a concentration-dependent manner. However, fluconazole had no effect on mepivacaine-induced contraction. PD 98059, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, and indomethacin attenuated mepivacaine-induced ERK phosphorylation. Mepivacaine upregulated 5-LOX and COX-2 expression. These results suggest that mepivacaine-induced contraction involves ERK activation, which is primarily mediated by the 5-LOX pathway and in part by the COX-2 pathway.

  5. Hepatocyte Growth Factor Inhibits Apoptosis by the Profibrotic Factor Angiotensin II via Extracellular Signal-regulated Kinase 1/2 in Endothelial Cells and Tissue Explants

    Science.gov (United States)

    2010-12-01

    II via Extracellular Signal-regulated Kinase 1/2 in Endothelial Cells and Tissue Explants Young H. Lee, Ana P. Marquez , Ognoon Mungunsukh, and Regina...L., Gonzalez- Garcia , M., Page, C., Herrera, R., and Nunez, G. (1997). Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt... Marquez , A. P., and Day, R. M. (2010). Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial

  6. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  7. Neuronal nitric oxide contributes to neuroplasticity-associated protein expression through cGMP, protein kinase G, and extracellular signal-regulated kinase.

    Science.gov (United States)

    Gallo, Eduardo F; Iadecola, Costantino

    2011-05-11

    Nitric oxide (NO) synthesized by neuronal NO synthase (nNOS) has long been implicated in brain plasticity. However, it is unclear how this short-lived mediator contributes to the long-term molecular changes underlying neuroplasticity, which typically require activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway and gene expression. To address this issue, we used a neuroplasticity model based on treatment of neuronal cultures with bicuculline and a model of experience-dependent plasticity in the barrel cortex. In neuronal cultures, NOS inhibition attenuated the bicuculline-induced activation of ERK and the expression of c-Fos, Egr-1, Arc, and brain-derived neurotrophic factor (BDNF), proteins essential for neuroplasticity. Furthermore, inhibition of the NO target soluble guanylyl cyclase or of the cGMP effector kinase protein kinase G (PKG) reduced both ERK activation and plasticity-related protein expression. NOS inhibition did not affect phosphorylation of cAMP response element-binding protein (CREB), a well-established ERK nuclear target, but it attenuated the nuclear accumulation of the CREB coactivator TORC1 and suppressed the activation of Elk-1, another transcription factor target of ERK. Consistent with these in vitro observations, induction of c-Fos, Egr-1, and BDNF was attenuated in the D1 cortical barrel of nNOS(-/-) mice subjected to single whisker experience. These results establish nNOS-derived NO as a key factor in the expression of proteins involved in neuroplasticity, an effect mediated through cGMP, PKG, and ERK signaling. These actions of NO do not depend on CREB phosphorylation but may involve TORC1 and Elk-1. Our data unveil a previously unrecognized link between neuronal NO and the molecular machinery responsible for the sustained synaptic changes underlying neuroplasticity.

  8. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

    Science.gov (United States)

    Hara, Tomoya; Kakudo, Natsuko; Morimoto, Naoki; Ogawa, Takeshi; Lai, Fangyuan; Kusumoto, Kenji

    2016-12-01

    Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

  9. OVER-EXPRESSION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN VASCULAR SMOOTH MUSCLE CELL OF HYPERTENSIVE RATS

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension. Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery. The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively. The control group were sham operated age-matched Wistar rats. Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats. Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00 ± 33.00 mm Hg at the end of experiment, significantly higher than that in the control rats ( P < 0. 01 ). Blood pressure in SHR4w ( 108.00 ± 11.25 mm Hg) was similar to that in the controls. However, it rose to 122.25 ± 21.75 mm Hg in SHR8w, and even up to 201.75 ± 18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls ( P < 0. 01 ). The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P < 0. 05 ). Hyaline degeneration of the afferent arterioles was found in WHR. In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w,and SHR16w. Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2. The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7. 09% ± 1.75%, 14. 57% ± 4. 58%, 29.44% ± 7. 35%, and 13.63% ±3.85%, respectively) than that of the controls( P < 0. 01 ). The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR

  10. Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

    Institute of Scientific and Technical Information of China (English)

    Zhuoxin Yang; Lihong Diao; Haibo Yu; Wenshu Luo; Ling Wang; Min Pi; Xiaodan Rao; Junhua Peng

    2008-01-01

    BACKGROUND: The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells.OBJECTIVE: To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention.DESIGN: Randomized controlled study.SETTING: Department of Anatomy, Sun Yat-Sen University. MATERIALS: A total of 60 healthy adult male Wistar rats weighing (250±10) g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS: The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups: normal group (n = 6), sham operation group (n = 18), model group (n = 18), and electroacupuncture group (n = 18). Middle cerebral artery occlusion (MCAO) was performed in the model group and electroacupuncture group. Zea Longa's grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between 1 and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery (CCA) was isolated, and the external carotid artery (ECA) was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture (People's Publishing House; 1997; First Edition). The Chengjiang, Qihai, and Guanyuan

  11. The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway.

    Science.gov (United States)

    Park, Jong-In; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2003-01-01

    Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.

  12. Extracellular signal-regulated kinases 1/2 and Akt contribute to triclosan-stimulated proliferation of JB6 Cl 41-5a cells.

    Science.gov (United States)

    Wu, Yuanfeng; Beland, Frederick A; Chen, Si; Fang, Jia-Long

    2015-08-01

    Triclosan is a broad spectrum anti-bacterial agent widely used in many personal care products, household items, medical devices, and clinical settings. Human exposure to triclosan is mainly through oral and dermal routes. In previous studies, we found that sub-chronic dermal exposure of B6C3F1 mice to triclosan induced epidermal hyperplasia and focal necrosis; however, the mechanisms for these responses remain elusive. In this study, using mouse epidermis-derived JB6 Cl 41-5a cells, we found that triclosan stimulated cell growth in a concentration- and time-dependent manner. Enhanced cell proliferation was demonstrated by a substantial increase in the percentage of BrdU-positive cells, an elevation in the protein levels of cyclin D1 and cyclin A, and a reduction in the protein level of p27(Kip1). Western blotting analysis revealed that triclosan induced the activation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38, and Akt. Pre-treatment of the cells with PD184352, an inhibitor of the upstream kinase MEK1/2, or with wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked triclosan-mediated phosphorylation of ERK1/2 and Akt, respectively, and substantially suppressed triclosan-stimulated cell proliferation, whereas the JNK inhibitor SP600125 or the p38 inhibitor SB203580 had little to no effect on triclosan-stimulated cell proliferation. The phosphorylation activation of ERK1/2 and Akt was further confirmed on the skin of mice dermally administered triclosan. These data suggest that the activation of ERK1/2 and Akt is involved in triclosan-stimulated proliferation of JB6 Cl 41-5a cells.

  13. Orexin A modulates INS-1E cell proliferation and insulin secretion via extracellular signal-regulated kinase and transient receptor potential channels.

    Science.gov (United States)

    Skrzypski, M; Khajavi, N; Mergler, S; Billert, M; Szczepankiewicz, D; Wojciechowicz, T; Nowak, K W; Strowski, M Z

    2016-10-01

    Orexins A (OXA) and B (OXB) control energy homeostasis by regulating food intake, energy expenditure and sleep-wake cycle. Several studies showed that OXA stimulates insulin secretion and proliferation of beta cells. However, mechanisms of action are still not well understood. Here, we investigated whether ERK and transient receptor potential channels (TRPs) play a role in mediating the effect of OXA on cell growth, insulin production, and secretion using the established INS-1E cell line. Cell proliferation was measured using BrdU assay. Insulin mRNA expression was detected by real-time PCR. Insulin secretion was assessed using ELISA. Intracellular calcium levels were measured using fluorescence calcium imaging (fura-2/AM). Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was detected by Western blot. TRP channel activity was blocked by lanthanum (III) chloride (La(3+); 100 - 300 μM) or ruthenium red (RuR; 10 μM). OXA (100 nM) stimulated INS-1E cell proliferation, insulin secretion, intracellular Ca(2+) concentration and ERK1/2 phosphorylation, without changing insulin mRNA expression. Inhibition of ERK1/2 by 10 μM U0126 attenuated OXA-stimulated INS-1E cell proliferation. Blockade of TRP channel activity by La(3+) or RuR rendered OXA ineffective at modulating Ca(2+) regulation and insulin release. In contrast, the L-type channel blocker nifedipine (10 μM) failed to affect OXA-stimulated insulin release. Taken together, OXA increases INS-1E cell proliferation via ERK1/2-dependent mechanism. Furthermore, OXA stimulates insulin secretion from INS-1E cells. TRPs are relevant for OXA-stimulated insulin secretion and intracellular calcium regulation.

  14. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xining; Gritsenko, Marina A.; Zhao, Rui; Monroe, Matthew E.; Pertz, Olivier C.; Purvine, Samuel O.; Orton, Daniel J.; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2011-05-20

    Abstract - Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

  15. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction*

    Science.gov (United States)

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xinning; Gritsenko, Marina A.; Zhao, Rui; Monore, Matthew E.; Pertz, Olivier C.; Purvine, Samuel O.; Orton, Daniel J.; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2011-01-01

    Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis. PMID:21454597

  16. Spatial phosphoprotein profiling reveals a compartmentalized extracellular signal-regulated kinase switch governing neurite growth and retraction.

    Science.gov (United States)

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xinning; Gritsenko, Marina A; Zhao, Rui; Monore, Matthew E; Pertz, Olivier C; Purvine, Samuel O; Orton, Daniel J; Jacobs, Jon M; Camp, David G; Smith, Richard D; Klemke, Richard L

    2011-05-20

    Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

  17. The Ras/Raf/MEK/Extracellular Signal-Regulated Kinase Pathway Induces Autocrine-Paracrine Growth Inhibition via the Leukemia Inhibitory Factor/JAK/STAT Pathway

    OpenAIRE

    Park, Jong-In; Strock, Christopher J.; Ball, Douglas W.; Nelkin, Barry D.

    2003-01-01

    Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF express...

  18. Extracellular signal regulated kinases 1/2 signal pathway and responses of astrocytes after diffuse brain injury

    Institute of Scientific and Technical Information of China (English)

    Jinxing Li; Haimei Zhao; Yu Li; Chong Wang; Jiashan Zhao; Xianli Zhu

    2007-01-01

    BACKGROUND: The treatment of diffuse brain injury during an acute period is focused on relieving degrees of secondary brain injury. Generation and development of pathological changes of secondary brain injury depend on signal conduction, so down-regulating over response of astrocyte through interfering a key link of signal conduction pathway may bring a new thinking for the treatment of diffuse brain injury. OBJECTIVE: To observe the effect of over activity of extracellular signal regulated kinases 1/2 (ERK1/2) signal pathway on the response of astrocyte during an acute period of diffuse brain injury. DESIGN: Completely randomized grouping and controlled animal study.SETTINGS: Department of Neurosurgery, the Third Affiliated Hospital, Nanchang University; Department of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: A total of 158 healthy male SD rats, of 11 weeks old, weighing 320 - 370 g, were provided by Experimental Animal Faulty, Tongji Medical College, Huazhong University of Science and Technology. Rabbit-anti-phosphorylated ERK1/2 (pERKl/2) polyclonal antibody was provided by R&D Company; rabbit-anti-glial fibrillary acidic protein (GFAP) polyclonal antibody, SP immunohistochemical kit and horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG by Santa Cruz Company; specific inhibitor U0126 of ERK1/2 signal pathway by Alexis Company. METHODS: The experiment was carried out in the Laboratory of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from September 2004 to March 2006. ①Detection of pERKl/2 expression: A total of 110 rats were randomly divided into sham operation group (n =5), model group (n =35), high-dosage U0126 group (n =35) and low-dosage U0126 group (n =35). Rats in the sham operation group were only treated with incision of epicranium and fixation of backup plate, but not hit. Rats in the model group

  19. Electroacupuncture at PC6 (Neiguan Improves Extracellular Signal-Regulated Kinase Signaling Pathways Through the Regulation of Neuroendocrine Cytokines in Myocardial Hypertrophic Rats

    Directory of Open Access Journals (Sweden)

    Jia Li

    2012-01-01

    Full Text Available Electroacupuncture (EA therapy has been widely accepted as a useful therapeutic technique with low or no risk in the clinical prevention of cardiac hypertrophy. However, the signaling transduction mechanism underlying this effect remains unclear. The current study investigates the effects of EA on the signaling pathways of myocardial hypertrophy (MH in rats. Up to 40 3-month-old Sprague-Dawley (SD rats were randomly divided into normal, model, PC6 (Neiguan, and LI4 (Hegu groups, with ten rats in each group. All the rats except for the normal group received 3 mg/kg⋅d of isoprinosine hydrochloride (ISO injection into the back skin. The rats in the PC6 and LI4 groups received EA for 14 days. On the 15th day, electrocardiograms were recorded, and the ultrastructure of the myocardial cells was observed. The myocardial hypertrophy indices (MHIs, electrocardiograph (ECG, ultrastructure observation, levels of plasma angiotensin II (Ang II and endothelin (ET, as well as protein expression of extracellular signal-regulated kinase (ERK, and phosphorylation extracellular signal regulating kinase (p-ERK in the left ventricular myocardial tissue were measured. The results indicated that EA can improve cardiac function in MH rats by modulating upstream neuroendocrine cytokines that regulate the ERK signaling pathways.

  20. Region- or state-related differences in expression and activation of extracellular signal-regulated kinases (ERKs in naïve and pain-experiencing rats

    Directory of Open Access Journals (Sweden)

    Cui Xiu-Yu

    2007-07-01

    Full Text Available Abstract Background Extracellular signal-regulated kinase (ERK, one member of the mitogen-activated protein kinase (MAPK family, has been suggested to regulate a diverse array of cellular functions, including cell growth, differentiation, survival, as well as neuronal plasticity. Recent evidence indicates a role for ERKs in nociceptive processing in both dorsal root ganglion and spinal cord. However, little literature has been reported to examine the differential distribution and activation of ERK isoforms, ERK1 and ERK2, at different levels of pain-related pathways under both normal and pain states. In the present study, quantitative blot immunolabeling technique was used to determine the spatial and temporal expression of ERK1 and ERK2, as well as their activated forms, in the spinal cord, primary somatosensory cortex (SI area of cortex, and hippocampus under normal, transient pain and persistent pain states. Results In naïve rats, we detected regional differences in total expression of ERK1 and ERK2 across different areas. In the spinal cord, ERK1 was expressed more abundantly than ERK2, while in the SI area of cortex and hippocampus, there was a larger amount of ERK2 than ERK1. Moreover, phosphorylated ERK2 (pERK2, not phosphorylated ERK1 (pERK1, was normally expressed with a high level in the SI area and hippocampus, but both pERK1 and pERK2 were barely detectable in normal spinal cord. Intraplantar saline or bee venom injection, mimicking transient or persistent pain respectively, can equally initiate an intense and long-lasting activation of ERKs in all three areas examined. However, isoform-dependent differences existed among these areas, that is, pERK2 exhibited stronger response than pERK1 in the spinal cord, whereas ERK1 was more remarkably activated than ERK2 in the S1 area and hippocampus. Conclusion Taken these results together, we conclude that: (1 under normal state, while ERK immunoreactivity is broadly distributed in the rat

  1. Adenovirus-induced extracellular signal-regulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    during the late phase of infection. Pharmacologic inhibition of ERK phosphorylation reduced virus recovery by >100-fold. Blocking MEK/ERK signaling affected virus DNA replication and mRNA levels only weakly but strongly reduced the amount of viral proteins, independently of the kinases MNK1 and PKR....... Hence, adenovirus induces the oncogenic Raf/MEK/ERK signaling pathway to enhance viral progeny by sustaining the levels of viral proteins. Concerning therapy, our results suggest that the use of Raf/MEK/ERK inhibitors will interfere with the propagation of oncolytic adenoviruses.......The Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling cascade enhances tumor cell proliferation in many cases. Here, we show that adenovirus type 5, a small DNA tumor virus used in experimental cancer therapy, strongly induces ERK phosphorylation...

  2. Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells.

    Science.gov (United States)

    Lipschutz, Joshua H; Li, Shixiong; Arisco, Amy; Balkovetz, Daniel F

    2005-02-01

    The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.

  3. Light induces Fos expression via extracellular signal-regulated kinases 1/2 in melanopsin-expressing PC12 cells

    DEFF Research Database (Denmark)

    Moldrup, Marie-Louise Bülow; Georg, Birgitte; Falktoft, Birgitte

    2010-01-01

    The photopigment melanopsin is expressed in a subtype of mammalian ganglion cells in the retina that project to the circadian clock in the hypothalamic suprachiasmatic nucleus to mediate non-visual light information. Melanopsin renders these retinal ganglion cells intrinsically photosensitive and...

  4. Light induces Fos expression via extracellular signal-regulated kinases 1/2 in melanopsin-expressing PC12 cells

    DEFF Research Database (Denmark)

    Moldrup, Marie-Louise Bülow; Georg, Birgitte; Falktoft, Birgitte;

    2010-01-01

    The photopigment melanopsin is expressed in a subtype of mammalian ganglion cells in the retina that project to the circadian clock in the hypothalamic suprachiasmatic nucleus to mediate non-visual light information. Melanopsin renders these retinal ganglion cells intrinsically photosensitive and...

  5. Involvement of extracellular signal-regulated kinase/mitogen activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jian Guan; Xiao-Ping Chen; Hong Zhu; Shun-Feng Luo; Bin Cao; Lei Ding

    2004-01-01

    AIM: To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes:multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.METHODS: A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure.RESULTS: Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold).Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the

  6. Metformin inhibits aromatase via an ERK (extracellular signal-regulated kinase) - mediated pathway

    OpenAIRE

    Rice, Suman; Pellatt, Laura; Ramanathan, Kumaran; Whitehead, Saffron Anne; Mason, Helen Diane

    2009-01-01

    Metformin treatment, now widely prescribed in PCOS, is aimed at correcting the associated insulin resistance, but it has also been shown to directly inhibit ovarian steroidogenesis. The mechanisms however, by which metformin inhibits oestradiol production in human granulosa cells remain unknown. Granulosa luteal cells were incubated with metformin, insulin or combined metformin and insulin treatment and aromatase mRNA expression was quantified using real-time PCR. Enzyme activity was assessed...

  7. Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation.

    Science.gov (United States)

    Liu, Xiangyuan; Chen, Min; Li, Long; Gong, Liyan; Zhou, Hu; Gao, Daming

    2017-03-10

    Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Bing Yao; Hai-Yan Liu; Yu-Chun Gu; Shan-Shan Shi; Xiao-Qian Tao; Xiao-Jun Li; Yi-Feng Ge; Ying-Xia Cui; Guo-Bin Yang

    2011-01-01

    Gonadotropin-releasing hormone (GnRH) is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase (MAPK) pathways are involved in GnRH agonist (GnRHa)-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay (RIA). The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3β-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L-1 after 24 h, activation of ERK1/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERK1/2, the upregulation of 3β-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3β-HSDgene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Robbins, David J. [Department of Surgery, Molecular Oncology Program, Miller School of Medicine, University of Miami, Miami (United States); Matalon, Sadis [Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL (United States); Deshane, Jessy S. [Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Afaq, Farrukh [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Bickers, David R. [Department of Dermatology, Columbia University Medical Center, New York (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States)

    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.

  10. Carbonic anhydrase activation enhances object recognition memory in mice through phosphorylation of the extracellular signal-regulated kinase in the cortex and the hippocampus.

    Science.gov (United States)

    Canto de Souza, Lucas; Provensi, Gustavo; Vullo, Daniela; Carta, Fabrizio; Scozzafava, Andrea; Costa, Alessia; Schmidt, Scheila Daiane; Passani, Maria Beatrice; Supuran, Claudiu T; Blandina, Patrizio

    2017-05-15

    Rats injected with by d-phenylalanine, a carbonic anhydrase (CA) activator, enhanced spatial learning, whereas rats given acetazolamide, a CA inhibitor, exhibited impairments of fear memory consolidation. However, the related mechanisms are unclear. We investigated if CAs are involved in a non-spatial recognition memory task assessed using the object recognition test (ORT). Systemic administration of acetazolamide to male CD1 mice caused amnesia in the ORT and reduced CA activity in brain homogenates, while treatment with d-phenylalanine enhanced memory and increased CA activity. We provided also the first evidence that d-phenylalanine administration rapidly activated extracellular signal-regulated kinase (ERK) pathways, a critical step for memory formation, in the cortex and the hippocampus, two brain areas involved in memory processing. Effects elicited by d-phenylalanine were completely blunted by co-administration of acetazolamide, but not of 1-N-(4-sulfamoylphenyl-ethyl)-2,4,6-trimethylpyridinium perchlorate ((C18),) a CA inhibitor that, differently from acetazolamide, does not cross the blood brain barrier. Our results strongly suggest that brain but not peripheral CAs activation potentiates memory as a result of ERK pathway enhanced activation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction.

    Science.gov (United States)

    Vomastek, Tomás; Iwanicki, Marcin P; Burack, W Richard; Tiwari, Divya; Kumar, Devanand; Parsons, J Thomas; Weber, Michael J; Nandicoori, Vinay Kumar

    2008-11-01

    Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

  12. Short-term anesthesia inhibits formalin-induced extracellular signal-regulated kinase (ERK) activation in the rostral anterior cingulate cortex but not in the spinal cord.

    Science.gov (United States)

    Tochiki, Keri K; Maiarù, Maria; Miller, James R C; Hunt, Stephen P; Géranton, Sandrine M

    2015-08-14

    The rostral anterior cingulate cortex (rACC) has been implicated in the negative affective response to injury, and importantly, it has been shown that activation of extracellular signal-regulated kinase (ERK) signaling in the rACC contributes to the full expression of the affective component of pain in rodents. In this study, we investigated whether administration of anesthesia at the time of injury could reduce phosphorylated-ERK (PERK) expression in the rACC, which might eliminate the negative affective component of noxious stimulation. Intraplantar hindpaw formalin stimulation, an aversive event in the awake animal, was given with or without general isoflurane anesthesia, and PERK expression was subsequently quantified in the rACC using immunohistochemistry. Furthermore, as numerous studies have demonstrated the importance of spinal ERK signaling in the regulation of nociceptive behaviour, we also examined PERK in the superficial dorsal horn of the spinal cord. Formalin injection with and without short-term (anesthesia induced the same level of PERK expression in spinal cord laminae I-II. However, PERK expression was significantly inhibited across all laminae of the rACC in animals anesthetized during formalin injection. The effect of anesthesia was such that levels of PERK were the same in formalin and sham treated anesthesized animals. This study is the first to demonstrate that isoflurane anesthesia can inhibit formalin-induced PERK in the rACC and therefore might eliminate the unpleasantness of restraint associated with awake hindpaw injection.

  13. Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages.

    Science.gov (United States)

    Chen, Renqiong; Li, Xiqing; Lu, Sha; Ma, Tuan; Huang, Xiaowen; Mylonakis, Eleftherios; Liang, Yuheng; Xi, Liyan

    2014-05-01

    Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome-lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.

  14. Role of crosstalk between phosphatidylinositol 3-kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways in artery-vein specification.

    Science.gov (United States)

    Hong, Charles C; Kume, Tsutomu; Peterson, Randall T

    2008-09-12

    Functional and structural differences between arteries and veins lie at the core of the circulatory system, both in health and disease. Therefore, understanding how artery and vein cell identities are established is a fundamental biological challenge with significant clinical implications. Molecular genetic studies in zebrafish and other vertebrates in the past decade have begun to reveal in detail the complex network of molecular pathways that specify artery and vein cell fates during embryonic development. Recently, a chemical genetic approach has revealed evidence that artery-vein specification is governed by cross talk between phosphoinositide 3-kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in artery-vein specification. We discuss recent findings on the signaling pathways involved in artery-vein specification during zebrafish development and compare and contrast these results to those from mammalian systems. It is anticipated that the complementary approaches of genetics and chemical biology, involving a variety of model organisms and systems, will lead to a better understanding of artery-vein specification and possibly to novel therapeutic approaches to treat vascular diseases.

  15. Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation▿ †

    Science.gov (United States)

    Artero-Castro, Ana; Callejas, Francisco B.; Castellvi, Josep; Kondoh, Hiroshi; Carnero, Amancio; Fernández-Marcos, Pablo J.; Serrano, Manuel; Ramón y Cajal, Santiago; Lleonart, Matilde E.

    2009-01-01

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research. PMID:19158277

  16. BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

    Science.gov (United States)

    Oyama, Satomi; Funasaka, Yoko; Watanabe, Atsushi; Takizawa, Toshihiro; Kawana, Seiji; Saeki, Hidehisa

    2015-05-01

    To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma.

  17. Dynamic regulation of extracellular signal-regulated kinase (ERK by protein phosphatase 2A regulatory subunit B56γ1 in nuclei induces cell migration.

    Directory of Open Access Journals (Sweden)

    Ei Kawahara

    Full Text Available Extracellular signal-regulated kinase (ERK signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.

  18. Study on Effects of Extracts from Salvia Miltiorrhiza and Curcuma Longa in Inhibiting Phosphorylated Extracellular Signal Regulated Kinase Expression in Rat's Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    CHENG Yang; PING Jian; LIU Cheng; TAN Ying-zi; CHEN Gao-feng

    2006-01-01

    Objective: To study the effect of salvianolic acid B (SAB) and curcumin, the extracts of Salvia Miltiorrhiza and Curcuma Longa, on the proliferation and activation of hepatic stellate cell (HSC), and the extracellular signal regulated kinase (ERK) expression in it. Methods: Rat's HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-(4,5-dimthyl-2-2thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and the expression levels of α smooth actin (α-SMA), collagen type Ⅰ , and ERK were determined by Western blot. Results: SAB and curcumin inhibited the proliferation and activation of rat's HSC-T6 in dose-dependent fashion and significantly reduced the expression level of α-SMA ( P<0.01 ). Curcumin significantly reduced the expression of collagen type Ⅰ( P<0.05). Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group ( P<0.01 and P<0.05 respectively). Conclusion: SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.

  19. Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    WU Zhen; WU Li-jun; TASHIRO Shinichi; ONODERA Satoshi; IKEJIMA Takashi

    2005-01-01

    Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin.Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4’,6’-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

  20. Matrine-induced apoptosis of human nasopharyngeal carcinoma cells via in vitro vascular endothelial growth factor-A/extracellular signal-regulated kinase1/2 pathway inactivation.

    Science.gov (United States)

    Xie, M; He, G; Wang, R; Shi, S; Chen, J; Ye, Y; Xie, L; Yi, X; Tang, A

    2014-07-01

    Matrine, a main active extract from Sophora flavescens Ait, has been demonstrated to exert anticancer effects on various cancer cell lines, such as malignant melanoma, breast cancer, and lung cancer. However, it is currently unclear whether matrine could also elicit an inhibitory effect on growth of nasopharyngeal carcinoma (NPC), let alone the possible molecular mechanisms. Therefore, in a previous study, we investigated matrine-induced proliferation inhibition and apoptosis in NPC cells. It was shown that proliferation of human NPC cells (CNE1 and CNE2) was significantly diminished by matrine in a dose- and time-dependent manner, and apoptosis was induced in both 2 NPC cells, particularly in CNE2 cells. Moreover, the increased apoptosis rate in matrine-treated CNE2 cells confirmed the proapoptotic activity of matrine. We further found that matrine treatment dose- and time-dependently reduced the levels of vascular endothelial growth factor-A (VEGF-A), and inactivated extracellular signal-regulated kinase1/2 (ERK1/2), followed by increased expression of downstream target caspase-3. Overall, we conclude that matrine could induce apoptosis of human NPC cells via VEGF-A/ERK1/2 pathway, which supports the potential use of matrine in clinically treating NPC.

  1. Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Weiguo, E-mail: liangweiguo@tom.com [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Fang, Dejian [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Ye, Dongping [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia); Zou, Longqiang; Shen, Yan; Dai, Libing [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Xu, Jiake, E-mail: jiake.xu@uwa.edu.au [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia)

    2014-07-11

    Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.

  2. Genetic interaction of two abscisic acid signaling regulators, HY5 and FIERY1, in mediating lateral root formation

    KAUST Repository

    Chen, Hao

    2011-01-01

    Root architecture is continuously shaped in a manner that helps plants to better adapt to the environment. Gene regulation at the transcriptional or post-transcriptional levels largely controls this environmental response. Recently, RNA silencing has emerged as an important player in gene regulation and is involved in many aspects of plant development, including lateral root formation. In a recent study, we found that FIERY1, a bifunctional abiotic stress and abscisic acid (ABA) signaling regulator and an endogenous RNA silencing suppressor, mediates auxin response during lateral root formation in Arabidopsis. We proposed that FRY1 regulates lateral root development through its activity on adenosine 3,5-bisphosphate (PAP), a strong inhibitor of exoribonucleases (XRNs). Interestingly, some of the phenotypes of fry1, such as enhanced response to light in repressing hypocotyl elongation and hypersensitivity to ABA in lateral root growth, are opposite to those of another light- and ABA-signaling mutant, hy5. Here we analyzed the hy5 fry1 double mutant for root and hypocotyl growth. We found that the hy5 mutation can suppress the enhanced light sensitivity in fry1 hypocotyl elongation and restore the lateral root formation. The genetic interaction between HY5 and FRY1 indicates that HY5 and FRY1 may act in overlapping pathways that mediate light signaling and lateral root development. © 2011 Landes Bioscience.

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

    Institute of Scientific and Technical Information of China (English)

    Deshu Lin; Huibo Ren; Ying Fu

    2015-01-01

    In multicel ular plant organs, cel shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cel‐to‐cel communi-cation. Plants have a specific subfamily of the Rho GTPase family, usual y cal ed Rho of Plants (ROP), which serve as a critical signal transducer involved in many cel ular processes. In the last decade, important advances in the ROP‐mediated regulation of plant cel morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cel s. Especial y, the auxin‐ROP signaling networks have been demonstrated to control interdigitated growth of pavement cel s to form jigsaw‐puzzle shapes. Here, we review findings related to the discovery of this novel auxin‐signaling mecha-nism at the cel surface. This signaling pathway is to a large extent independent of the wel‐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 al owing ROP proteins to serve as a bustling signal decoder and integrator for plant cel morphogenesis.

  4. β-Hydroxybutyric acid inhibits growth hormone-releasing hormone synthesis and secretion through the GPR109A/extracellular signal-regulated 1/2 signalling pathway in the hypothalamus.

    Science.gov (United States)

    Fu, S-P; Liu, B-R; Wang, J-F; Xue, W-J; Liu, H-M; Zeng, Y-L; Huang, B-X; Li, S-N; Lv, Q-K; Wang, W; Liu, J-X

    2015-03-01

    β-Hydroxybutyric acid (BHBA) has recently been shown to regulate hormone synthesis and secretion in the hypothalamus. However, little is known about the effects of BHBA-mediated hormone regulation or the detailed mechanisms by which BHBA regulates growth hormone-releasing hormone (GHRH) synthesis and secretion. In the present study, we examined the expression of the BHBA receptor GPR109A in primary hypothalamic cell cultures. We hypothesised that BHBA regulates GHRH via GPR109A and its downstream signals. Initial in vivo studies conducted in rats demonstrated that GHRH mRNA expression in the hypothalamus was strongly inversely correlated with BHBA levels in the cerebrospinal fluid during postnatal development (r = -0.89, P hypothalamus in both in vivo and in vitro, and this effect was also inhibited by PTX in vitro. In primary hypothalamic cells, BHBA activated the extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) kinases, as shown by western blot analysis. Moreover, inhibition of ERK1/2 with U0126 attenuated the BHBA-mediated reduction in Gsh-1 expression and GHRH synthesis and secretion. These results strongly suggest that BHBA directly regulates GHRH synthesis and secretion via the GPR109A/ERK1/2 MAPK pathway, and also that Gsh-1 is essential for this function. © 2015 British Society for Neuroendocrinology.

  5. Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages.

    Science.gov (United States)

    Shikata, Yayoi; Okada, Toshiaki; Hashimoto, Mitsuhiro; Ellis, Tammy; Matsumaru, Daisuke; Shiroishi, Toshihiko; Ogawa, Masaharu; Wainwright, Brandon; Motoyama, Jun

    2011-01-15

    Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5-18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.

  6. Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

    Directory of Open Access Journals (Sweden)

    Kirk Ruth S

    2009-04-01

    Full Text Available Abstract Background Schistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs are released. Nitric oxide (NO and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK phosphorylation (activation in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated. Results Haemocytes from resistant snails challenged with S. mansoni ESPs (20 μg/ml over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1–10 μg/ml did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1–20 μg/ml. Western blotting revealed that U0126 (1 μM or 10 μM blocked the phosphorylation (activation status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells. Conclusion S. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host.

  7. Extracellular signal-regulated protein kinase activation in spinal cord contributes to pain hypersensitivity in a mouse model of type 2 diabetes.

    Science.gov (United States)

    Xu, Xiang; Chen, Hui; Ling, Bing-Yu; Xu, Lan; Cao, Hong; Zhang, Yu-Qiu

    2014-02-01

    Painful peripheral neuropathy is a common complication of diabetes mellitus. The symptom of pain can become a major factor that decreases the quality of life of patients with diabetes, while effective treatment is lacking. In the present study, we aimed to investigate the changes of pain threshold in the early stage of diabetes in db/db mice, an animal model of type 2 diabetes mellitus, and the underlying molecular mechanisms. We found that (1) db/db mice (with a leptin receptor-null mutation and characterized by obesity and hyperglycemia) showed hypersensitivity to mechanical and thermal stimuli at the early stage of diabetes; (2) phosphorylated extracellular signal-regulated kinase (pERK), but not total ERK in the spinal cord and dorsal root ganglia in db/db mice significantly increased compared with wild-type mice. The increased pERK immunoreactivity occurred in both NeuN-expressing neurons and GFAP-expressing astrocytes, but not in Iba-1-expressing microglia; (3) both single and consecutive (for 5 days) intrathecal injections of U0126 (2 nmol per day), a selective MEK (an ERK kinase) inhibitor beginning at 8 weeks of age, attenuated the bilateral mechanical allodynia in the von-Frey test and heat hyperalgesia in Hargreave's test; and (4) db/db mice also displayed increased nocifensive behavior during the formalin test, and this was blocked by intrathecal injection of U0126. Also, the expression of pERK1 and pERK2 was upregulated following the formalin injection. Our results suggested that the activation of ERK in spinal neurons and astrocytes is correlated with pain hypersensitivity of the type 2 diabetes animal model. Inhibiting the ERK pathway may provide a new therapy for pain control in type 2 diabetes.

  8. Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signalling in a High-Throughput Format

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAyoub

    2014-06-01

    Full Text Available The extracellular signal-regulated kinases (ERKs are key components of multiple important cell signalling pathways regulating diverse biological responses. This signalling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs and receptor tyrosine kinases (RTKs. We report the development of a new cell-based phospho-ERK1/2 assay (designated Phospho-ERK, which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor (EGFR as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently or stably expressing the different receptors. The validation was performed for agonists, antagonists and inhibitors in dose-response as well as kinetic analysis, and the signalling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z’-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signalling. Finally, our study is of great interest in the current context of investigating ERK1/2 signalling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signalling.

  9. Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

    Institute of Scientific and Technical Information of China (English)

    SHI Zhong Fang; ZHAO Wei Jiang; XU Li Xin; DONG Li Ping; YANG Shao Hua; YUAN Fang

    2015-01-01

    ObjectiveTo investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in the regulation of aquaporin 4 (AQP4) expression inculturedastrocytes after scratch-injury. MethodsThe scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase (LDH) leakages were observed to assess the degree of scratch-injury. AQP4 expressionwas detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2 (p-ERK1/2) expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 µmol/L U0126 (ERK1/2inhibitor) was incubated in the medium at 30 min before the scratch-injury in some groups. ResultsIncreases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expressionwas associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. ConclusionThese results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

  10. Inflammation in the uterus induces phosphorylated extracellular signal-regulated kinase and substance P immunoreactivity in dorsal root ganglia neurons innervating both uterus and colon in rats.

    Science.gov (United States)

    Li, Jichang; Micevych, Paul; McDonald, John; Rapkin, Andrea; Chaban, Victor

    2008-09-01

    In women, clinical studies suggest that pain syndromes such as irritable bowel syndrome and interstitial cystitis, which are associated with visceral hyperalgesia, are often comorbid with endometriosis and chronic pelvic pain. One of the possible explanations for this phenomenon is viscerovisceral cross-sensitization, in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input to the same dorsal root ganglion (DRG) from an unaffected visceral organ. Nociception induces up-regulation of cellular mechanisms such as phosphorylated extracellular signal-regulated kinase (pERK) and substance P (SP), neurotransmitters associated with induced pain sensation. The purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of pERK- and SP-positive neurons that received input from both the uterus and the colon. Cell bodies of colonic and uterine DRG were retrogradely labeled with fluorescent tracer dyes microinjected into the colon/rectum and into the uterus. Ganglia were harvested for fluorescent microscopy to identify positively stained neurons. Approximately 6% of neurons were colon specific and 10% uterus specific. Among these uterus- or colon-specific neurons, up to 3-5% of DRG neurons in the lumbosacral neurons (L1-S3 levels) received input from both visceral organs. Uterine inflammation increased the number of pERK- and SP-immunoreactive DRG neurons innervating specifically colon, or innervating specifically uterus, and those innervating both organs. These results suggest that a localized inflammation activates primary visceral afferents, regardless of whether they innervate the affected organ. This visceral sensory integration in the DRG may underlie the observed comorbidity of female pelvic pain syndromes.

  11. Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Kai WANG; Min ZHANG; Ying-ying QIAN; Zhe-yuan DING; Jun-huiLV; Hua-hao SHEN

    2011-01-01

    Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK1/2in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK1/2 or ERK1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

  12. Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum.

    Science.gov (United States)

    Yu, Hyun Sook; Kim, Se Hyun; Park, Hong Geun; Kim, Yong Sik; Ahn, Yong Min

    2011-11-01

    Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000μM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000μM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000μM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model.

  13. Chrysophanic acid reduces testosterone-induced benign prostatic hyperplasia in rats by suppressing 5α-reductase and extracellular signal-regulated kinase.

    Science.gov (United States)

    Youn, Dong-Hyun; Park, Jinbong; Kim, Hye-Lin; Jung, Yunu; Kang, JongWook; Jeong, Mi-Young; Sethi, Gautam; Seok Ahn, Kwang; Um, Jae-Young

    2017-02-07

    Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in male population, of which incidence increases gradually with age. In this study, we investigated the effect of chrysophanic acid (CA) on BPH. BPH was induced by a 4-week injection of testosterone propionate (TP). Four weeks of further injection with vehicle, TP, TP + CA, TP + finasteride was carried on. In the CA treatment group, the prostate weight was reduced and the TP-induced histological changes were restored as the normal control group. CA treatment suppressed the TP-elevated prostate specific antigen (PSA) expression. In addition, 5α-reductase, a crucial factor in BPH development, was suppressed to the normal level close to the control group by CA treatment. The elevated expressions of androgen receptor (AR), estrogen receptor α and steroid receptor coactivator 1 by TP administration were also inhibited in the CA group when compared to the TP-induced BPH group. Then we evaluated the changes in three major factors of the mitogen-activated protein kinase chain during prostatic hyperplasia; extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). While ERK was elevated in the process of BPH, JNK and p38 was not changed. This up-regulated ERK was also reduced as normal by CA treatment. Further in vitro studies with RWPE-1 cells confirmed TP-induced proliferation and elevated AR, PSA and p-ERK were all reduced by CA treatment. Overall, these results suggest a potential pharmaceutical feature of CA in the treatment of BPH.

  14. Roles of extracellular signal-regulated kinase 1/2 on the suppression of myostatin gene expression induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    Huazhoag Liu; Xiaorong An; Yongfu Chen; Jieping Zhong

    2008-01-01

    Basic fibmblast growth factor (bFGF, FG F-2 ) has an inhibitory effect on the expression of the myostatin gene in murine C2C12 myoblasts, as shown in our recent investigation. To further verify the regulatory effects of bFGF on the myostalin gene and to better understand its mechanism in skeletal muscle, and to promote clinical applications of bFGF to treat skeletal muscle diseases correlated to muscular dystrophy or AIDS and so on, recombinant human bFGF (rh-bFGF) was added into media and stimulated murine C2C12 myoblasts to investigate the dose-dependent effect ofbFGF on suppression of myostatin gene expression and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulatory mechanism. Simultaneously, complete coding sequence of ovine 18 kDa-bFGF gene was inserted into eukaryotic vector pCMV-neo (originated from pEGFP-N1 vector, from which the EGFP gene has been removed), the recombinant plasmid pCMV-neo-bFGF was harvested and injected into the mouse skeletal muscle of posterior limb. Expression levels of bFGF,myostatin, and ERKI/2 genes in murine C2C12 myoblasts and the skeletal muscle were analyzed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. The results showed that bFGFimpaired the expression ofmyostatin gene in a dose-dependent manner in C2C12 cells, with increasing concentration of rh-bFGF,myostatin mRNA declined gradually. In addition, results in skeletal muscle indicated that bFGF also suppressed the expression of the myostatin gene in vivo. Furthermore, we found ERKI/2 participated in the regulatory mechanism of bFGF on the expression of the myostatin gene.

  15. Acquisition and expression of Conditioned Taste Aversion differentially affects Extracellular signal Regulated Kinase and Glutamate receptor phosphorylation in rat Prefrontal Cortex and Nucleus Accumbens

    Directory of Open Access Journals (Sweden)

    Roberto eMarotta

    2014-05-01

    Full Text Available Conditioned taste aversion (CTA can be applied to study associative learning and its relevant underpinning molecular mechanisms in discrete brain regions. The present study examined, by immunohistochemistry and immunocytochemistry, the effects of acquisition and expression of lithium-induced CTA on activated Extracellular signal Regulated Kinase (p-ERK in the prefrontal cortex (PFCx and nucleus accumbens (Acb of male Sprague-Dawley rats. The study also examined, by immunoblotting, whether acquisition and expression of lithium-induced CTA resulted in modified levels of phosphorylation of glutamate receptor subunits (NR1 and GluR1 and Thr34- and Thr75-Dopamine-and-cAMP-Regulated PhosphoProtein (DARPP-32. CTA acquisition was associated with an increase of p-ERK-positive neurons and phosphorylated NR1 receptor subunit (p-NR1 in the PFCx, whereas p-GluR1, p-Thr34- and p-Thr75-DARPP-32 levels were not changed in this brain region. CTA expression increased the number of p-ERK-positive neurons in the shell (AcbSh and core (AcbC but left unmodified p-NR1, p-GluR1, p-Thr34- and p-Thr75-DARPP-32 levels. Furthermore, post-embedding immunogold quantitative analysis in AcbSh revealed that CTA expression significantly increased nuclear p-ERK immunostaining as well as p-ERK-labeled axo-spinous contacts. Overall, these results indicate that ERK and NR1, but not GluR1 and DARPP-32, are differentially phosphorylated as a consequence of acquisition and expression of aversive associative learning. Moreover, these results confirm that CTA represents an useful approach to study the molecular basis of associative learning in rats and suggest the involvement of ERK cascade in learning-associated synaptic plasticity.

  16. Effect of Sodium Tanshinone Ⅱ A Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinase1/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Shu-sheng; FENG Jun; ZHENG Zhi; LIANG Qian-sheng

    2008-01-01

    Objective:To observe the effects of sodium tanshinone Ⅱ A sulfonate(STS)on angiotensin Ⅱ(Ang Ⅱ)-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase(P-ERK1/2).Methods:In the primary culture of neonatal rat myocardial cells.the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by[3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells.The expression of p-ERK1/2 was determined using Western blot and immunofluorescence Iabeling.Results:(1)The totaI protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ (1 μmol/L)for 24 h;STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein.(2)After pretreatment of myocardial cells with Ang Ⅱ(1 μ mol/L)for 5 min,the p-ERK1/2 protein expression was increased,with the most obvious effect shown at about 10 min;pretreatment of myocardial cells with STS at different doses(2,10,50 μ mol/L)for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner.(3)After the myocardial cells were stimulated by Ang Ⅱ(1 μ mol/L),the immunofluorescence of ERK1/2 rapidly appeared in the nucleus.The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS.Conclusion:STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ,and the mechanism may be associated with the inhibition of p-ERK1/2 expression.

  17. Corticotrigeminal Projections from the Insular Cortex to the Trigeminal Caudal Subnucleus Regulate Orofacial Pain after Nerve Injury via Extracellular Signal-Regulated Kinase Activation in Insular Cortex Neurons.

    Science.gov (United States)

    Wang, Jian; Li, Zhi-Hua; Feng, Ban; Zhang, Ting; Zhang, Han; Li, Hui; Chen, Tao; Cui, Jing; Zang, Wei-Dong; Li, Yun-Qing

    2015-01-01

    Cortical neuroplasticity alterations are implicated in the pathophysiology of chronic orofacial pain. However, the relationship between critical cortex excitability and orofacial pain maintenance has not been fully elucidated. We recently demonstrated a top-down corticospinal descending pain modulation pathway from the anterior cingulate cortex (ACC) to the spinal dorsal horn that could directly regulate nociceptive transmission. Thus, we aimed to investigate possible corticotrigeminal connections that directly influence orofacial nociception in rats. Infraorbital nerve chronic constriction injury (IoN-CCI) induced significant orofacial nociceptive behaviors as well as pain-related negative emotions such as anxiety/depression in rats. By combining retrograde and anterograde tract tracing, we found powerful evidence that the trigeminal caudal subnucleus (Vc), especially the superficial laminae (I/II), received direct descending projections from granular and dysgranular parts of the insular cortex (IC). Extracellular signal-regulated kinase (ERK), an important signaling molecule involved in neuroplasticity, was significantly activated in the IC following IoN-CCI. Moreover, in IC slices from IoN-CCI rats, U0126, an inhibitor of ERK activation, decreased both the amplitude and the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and reduced the paired-pulse ratio (PPR) of Vc-projecting neurons. Additionally, U0126 also reduced the number of action potentials in the Vc-projecting neurons. Finally, intra-IC infusion of U0126 obviously decreased Fos expression in the Vc, accompanied by the alleviation of both nociceptive behavior and negative emotions. Thus, the corticotrigeminal descending pathway from the IC to the Vc could directly regulate orofacial pain, and ERK deactivation in the IC could effectively alleviate neuropathic pain as well as pain-related negative emotions in IoN-CCI rats, probably through this top-down pathway. These findings may help

  18. Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein*

    Science.gov (United States)

    Takahashi-Niki, Kazuko; Kato-Ose, Izumi; Murata, Hiroaki; Maita, Hiroshi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2015-01-01

    DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf. PMID:26048984

  19. Inhibition of Adipocyte Differentiation by Phytoestrogen Genistein Through a Potential Downregulation of Extracellular Signal-Regulated Kinases 1/2 Activity

    Science.gov (United States)

    Liao, Qing-Chuan; Li, Ya-Lin; Qin, Yan-Fang; Quarles, L. Darryl; Xu, Kang-Kang; Li, Rong; Zhou, Hong-Hao; Xiao, Zhou-Sheng

    2016-01-01

    In the current study, we investigated the effects of genistein on adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures and its potential signaling pathway. The terminal adipogenic differentiation was assessed by western-blotting analysis of adipogenic-specific proteins such as PPARγ, C/EBPα, and aP2 and the formation of adipocytes. Treatment of mouse BMSC cultures with adipogenic cocktail resulted in sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, at the early phase of adipogenesis (from days 3 to 9). Inhibition of ERK1/2 activation by PD98059, a specific MEK inhibitor, reversed the induced adipogenic differentiation. Genistein dose-dependently decreased the phosphorylation of ERK1/2 in mouse BMSC cultures. Genistein incubation for the entire culture period, as well as that applied during the early phase of the culture period, significantly inhibited the adipogenic differentiation of mouse BMSC cultures. While genistein was incubated at the late stage (after day 9), no inhibitory effect on adipogenic differentiation was observed. BMSC cultures treated with genistein in the presence of fibroblast growth factor-2 (FGF-2), an activator of the ERK1/2 signaling pathway, expressed normal levels of ERK1/2 activity, and, in so doing, are capable of undergoing adipogenesis. Our results suggest that activation of the ERK1/2 signaling pathway during the early phase of adipogenesis (from days 3 to 9) is essential to adipogenic differentiation of BMSC cultures, and that genistein inhibits the adipogenic differentiation through a potential downregulation of ERK1/2 activity at this early phase of adipogenesis. PMID:18384126

  20. Cafestol, a coffee-specific diterpene, is a novel extracellular signal-regulated kinase inhibitor with AP-1-targeted inhibition of prostaglandin E2 production in lipopolysaccharide-activated macrophages.

    Science.gov (United States)

    Shen, Ting; Lee, Jaehwi; Lee, Eunji; Kim, Seong Hwan; Kim, Tae Woong; Cho, Jae Youl

    2010-01-01

    Coffee is a popular beverage worldwide with various nutritional benefits. Diterpene cafestol, one of the major components of coffee, contributes to its beneficial effects through various biological activities such as chemopreventive, antitumorigenic, hepatoprotective, antioxidative and antiinflammatory effects. In this study, we examined the precise molecular mechanism of the antiinflammatory activity of cafestol in terms of prostaglandin E(2) (PGE(2)) production, a critical factor involved in inflammatory responses. Cafestol inhibited both PGE(2) production and the mRNA expression of cyclooxygenase (COX)-2 from lipopolysaccharide (LPS)-treated RAW264.7 cells. Interestingly, this compound strongly decreased the translocation of c-Jun into the nucleus and AP-1 mediated luciferase activity. In kinase assays using purified extracellular signal-regulated kinase 2 (ERK2) or immunoprecipitated ERK prepared from LPS-treated cells in the presence or absence of cafestol, it was found that this compound can act as an inhibitor of ERK2 but not of ERK1 and mitogen-activated protein kinase kinase 1 (MEK 1). Therefore our data suggest that cafestol may be a novel ERK inhibitor with AP-1-targeted inhibitory activity against PGE(2) production in LPS-activated RAW264.7 cells.

  1. A-raf and B-raf are dispensable for normal endochondral bone development, and parathyroid hormone-related peptide suppresses extracellular signal-regulated kinase activation in hypertrophic chondrocytes.

    Science.gov (United States)

    Provot, Sylvain; Nachtrab, Gregory; Paruch, Jennifer; Chen, Adele Pin; Silva, Alcino; Kronenberg, Henry M

    2008-01-01

    Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.

  2. Polycystin-1 Regulates Extracellular Signal-Regulated Kinase-Dependent Phosphorylation of Tuberin To Control Cell Size through mTOR and Its Downstream Effectors S6K and 4EBP1 ▿

    Science.gov (United States)

    Distefano, Gianfranco; Boca, Manila; Rowe, Isaline; Wodarczyk, Claas; Ma, Li; Piontek, Klaus B.; Germino, Gregory G.; Pandolfi, Pier Paolo; Boletta, Alessandra

    2009-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease characterized by bilateral renal cyst formation. Both hyperproliferation and hypertrophy have been previously observed in ADPKD kidneys. Polycystin-1 (PC-1), a large orphan receptor encoded by the PKD1 gene and mutated in 85% of all cases, is able to inhibit proliferation and apoptosis. Here we show that overexpression of PC-1 in renal epithelial cells inhibits cell growth (size) in a cell cycle-independent manner due to the downregulation of mTOR, S6K1, and 4EBP1. Upregulation of the same pathway leads to increased cell size, as found in mouse embryonic fibroblasts derived from Pkd1−/− mice. We show that PC-1 controls the mTOR pathway in a Tsc2-dependent manner, by inhibiting the extracellular signal-regulated kinase (ERK)-mediated phosphorylation of tuberin in Ser664. We provide a detailed molecular mechanism by which PC-1 can inhibit the mTOR pathway and regulate cell size. PMID:19255143

  3. Theobromine, the primary methylxanthine found in Theobroma cacao, prevents malignant glioblastoma proliferation by negatively regulating phosphodiesterase-4, extracellular signal-regulated kinase, Akt/mammalian target of rapamycin kinase, and nuclear factor-kappa B.

    Science.gov (United States)

    Sugimoto, Naotoshi; Miwa, Shinji; Hitomi, Yoshiaki; Nakamura, Hiroyuki; Tsuchiya, Hiroyuki; Yachie, Akihiro

    2014-01-01

    Theobromine, a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. We previously showed that methylxanthines, including caffeine and theophylline, have antitumor and antiinflammatory effects, which are in part mediated by their inhibition of phosphodiesterase (PDE). A member of the PDE family, PDE4, is widely expressed in and promotes the growth of glioblastoma, the most common type of brain tumor. The purpose of this study was to determine whether theobromine could exert growth inhibitory effects on U87-MG, a cell line derived from human malignant glioma. We show that theobromine treatment elevates intracellular cAMP levels and increases the activity of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, whereas it attenuates p44/42 extracellular signal-regulated kinase activity and the Akt/mammalian target of rapamycin kinase and nuclear factor-kappa B signal pathways. It also inhibits cell proliferation. These results suggest that foods and beverages containing cocoa bean extracts, including theobromine, might be extremely effective in preventing human glioblastoma.

  4. Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer's disease patients.

    Science.gov (United States)

    Esteras, Noemí; Alquézar, Carolina; Bermejo-Pareja, Félix; Bialopiotrowicz, Emilia; Wojda, Urszula; Martín-Requero, Angeles

    2013-04-01

    Previously, we reported a Ca(2+)/calmodulin (CaM)-dependent impairment of apoptosis induced by serum deprivation in Alzheimer's disease (AD) lymphoblasts. These cell lines showed downregulation of extracellular signal-regulated kinase (ERK)1/2 activity and elevated content of p21 compared with control cells. The aim of this study was to delineate the molecular mechanism underlying the distinct regulation of p21 content in AD cells. Quantitative reverse transcription polymerase chain reaction analysis demonstrated increased p21 messenger RNA (mRNA) levels in AD cells. The ERK1/2 inhibitor, PD98059, prevented death of control cells and enhanced p21 mRNA and protein levels. The CaM antagonist, calmidazolium, and the CaMKII inhibitor, KN-62, normalized the survival pattern of AD lymphoblasts by augmenting ERK1/2 activation and reducing p21 mRNA and protein levels. Upregulation of p21 transcription in AD cells appears to be the consequence of increased activity of forkhead box O3a (FOXO3a) as the result of diminished ERK1/2-mediated phosphorylation of this transcription factor, which in turn facilitates its nuclear accumulation. Murine double minute 2 (MDM2) protein levels were decreased in AD cells relative to control lymphoblasts, suggesting an impairment of FOXO3a degradation.

  5. The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways.

    Science.gov (United States)

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T; Portell, Craig A; Lannutti, Brian J; Almasan, Alexandru; Hsi, Eric D

    2013-10-01

    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

  6. 5-Hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells are extracellular signal-regulated kinase pathway depen-dent

    Institute of Scientific and Technical Information of China (English)

    Dan SONG; Huai-liang WANG; Shuang WANG; Xin-hua ZHANG

    2005-01-01

    Aim:To investigate the effect of 5-hydroxytryptamine transporter (5-HTT) inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN) to extracelluar signal regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. Methods: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection effi ciency was measured by observing the uptake of the fluorecein isothiocynate (FITC)-labeled antisense ODN in PASMCs. The effects of 5-HTT selective inhibi tor fluoxetine and ODNs on the proliferation of PASMCs were evaluated by cell number counting and cell cycle analysis, and measured by microculture tetrazo lium (MTT) assay and flow cytometry (FCM), respectively. Results: Liposomes mediated the transfection of ODNs into PASMCs with high efficiency. MTT assay showed fluoxetine (10 μmol/L, 1 μmol/L, and 100 nmol/L) concentration dependently inhibited the proliferation of PASMCs induced by 5-HT (1 μmol/L) in vitro. The proliferation rate of PASMCs by 5-HT was significantly inhibited by pretreatment with ERK1/2 antisense ODN (0.2 μmol/L) from 251%± 18% to 86%±5% (P<0.01). Flow cytometric analysis of cell cycle distribution showed that the increase of 5-HT induced S phase fraction (SPF) and proliferation index (PI) were significantly inhibited by fluoxetine (1 μmol/L) or antisense ODN with SPF from 36%±4% to 26%±3% and 24%±4%, and PI from 34%±2% to 29%±2% and 24%±2%,respectively. Conclusion: 5-HTT mediates the mitogenic effect of 5-HT on PASMCs and the proliferation of PASMCs induced by 5-HT is dependent on ERKs signal pathway.

  7. Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model

    Institute of Scientific and Technical Information of China (English)

    XIAO Jun; WANG Ke; FENG Yu-lin; CHEN Xue-rong; XU Dan; ZHANG Ming-ke

    2011-01-01

    Background Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease,which is closely associated with cigarette smoking.However,the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known.This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats.Methods A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks.Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126,a specific MEK1 kinase inhibitor,at doses of 0.25 mg/kg,0.5 mg/kg and 1 mg/kg for 14 days.Expression of MUC5AC mRNA and protein,ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR,immunohistochemistry and Western blotting.Results Cigarette smoke significantly increased airway goblet cells metaplasia,induced the overexpression of MUG5AC mRNA and protein in bronchial epithelia,and increased the ratio of p-ERK 1/2 and ERK 1/2.U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P <0.05).Moreover,there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P<0.05).Conclusions Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein.ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

  8. Effects of resistance exercise intensity on extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation in men.

    Science.gov (United States)

    Taylor, Lem W; Wilborn, Colin D; Kreider, Richard B; Willoughby, Darryn S

    2012-03-01

    Extracellular signal-regulated kinase (ERK) 1/2 signaling has been shown to be increased after heavy resistance exercise and suggested to play a role in the hypertrophic adaptations that are known to occur with training. However, the role that ERK1/2 may play in response to lower intensities of resistance exercise is unknown. Therefore, the purpose of this study was to determine the effects of resistance exercise intensity on ERK1/2 activity in human skeletal muscle. Twelve recreationally active men completed separate bouts of single-legged resistance exercise with 8-10 repetitions (reps) at 80-85% 1 repetition maximum (1RM) (85%) and 18-20 reps at 60-65% 1RM (65%) in a randomized crossover fashion. For both resistance exercise sessions, vastus lateralis biopsies and blood draws were taken immediately before exercise (PRE) and at 30 minutes (30MPST), 2 hours (2HRPST), and 6 hours (6HRPST) post exercise, with an additional blood draw occurring immediately after exercise (POST). The phosphorylated levels of pIGF-1R, pMEK1, pERK1/2, and activated Elk-1 were assessed by phosphoELISA, and serum insulin-like growth factor 1 (IGF-1) was assessed via enzyme-linked immunosorbent assay. Statistical analyses used a 2 × 4 (muscle responses) and 2 × 5 (serum responses) multivariate analysis of variance on delta values from baseline (p < 0.05). Both exercise intensities significantly increased the activity of insulin-like growth factor 1 receptor (IGF-1R), mitogen-activated protein kinase 1, ERK1/2, and Elk-1, with peak activity occurring at 2HRPST (p < 0.001). However, 65% resulted in a preferential increase in IGF-1R and Elk-1 activation when compared with 85% (p < 0.05). No differences were observed for serum IGF-1 levels regardless of intensity and time. These findings demonstrate that resistance exercise upregulates ERK1/2 signaling in a manner that does not appear to be preferentially dependent on exercise intensity.

  9. Sexual differences of the effects of prenatal stress on the expression of extracellular signal-regulated kinaseas in the hippocampus of offspring rats

    Institute of Scientific and Technical Information of China (English)

    Qing Cai; Zhongliang Zhu; Xiaoli Fan; Ning Jia; Qinghong Li; Liang Song; Hui Li; Jiankang Liu

    2006-01-01

    BACKGROUND: Prenatal stress has been shown to inhibit cell proliferation in the dentate gyrus and hippocampus, reduce hippocampal volume, and cause neuronal loss and oxidative damage in the hippocampus of offspring rats, but the sexual difference of the effects on offsprings is seldom referred to.OBJECTIVE: To observe the effect of prenatal stress to adult pregnant rats on expression of extracellular signal-regulated kinases (ERK) in hippocampus of the offspring rats of different genders.DESIGN : A randomized and control animal experiment.SETTING: Department of Physiology and Pathophysiology, School of Medicine, Xi'an Jiaotong University.MATERIALS: The experiments were carried out in the Key Laboratory of Environment and Gene Related Diseases (Xi'an Jiaotong University), Ministry of Education between October 2005 and March 2006. Fifteen female and five male adult Sprague-Dawley rats were adopted. Female rats weighing 230-250 g and male rats weighing 280-350 g were used.METHODS: The virgin female rats were placed overnight with adult male rats (3:1) for mating. A total of twelve pregnant rats were randomly assigned to prenatal stress group (PNS group, n=6) and control group (n=6). The pregnant rats of the PNS group were exposed to restraint stress on days 14-20 of pregnancy three times a day, 45 minutes for each time [9,13]. The restraint device was a transparent plastic tube (6.8 cm in diameter) with air holes for breathing and closed end. The length could be adjusted to accommodate the size of the animals. To prevent habituation of animals to the daily procedure, restraint periods were randomly shifted within certain time periods (8:00-11:00, 11:00-14:00, and 16:00-19:00). After birth,offsprings of all groups were culled to 8-10 litters in each group and housed in the same animal room, and kept together with their biologic mothers. The pregnant rats of the control group were left undisturbed. On day 21, after all the offspring were weaned, male and female pups

  10. Acupuncture combined with curcumin disrupts platelet-derived growth factor β receptor/extracellular signal-regulated kinase signalling and stimulates extracellular matrix degradation in carbon tetrachloride-induced hepatic fibrosis in rats.

    Science.gov (United States)

    Zhang, Xiao-Ping; Zhang, Feng; Zhang, Zi-Li; Ma, Jin; Kong, De-Song; Ni, Guang-Xia; Wang, Ai-Yun; Chen, Wen-Xing; Lu, Yin; Zheng, Shi-Zhong

    2012-12-01

    Acupuncture treatment has been increasingly used to treat chronic liver diseases. We previously reported that acupuncture combined with curcumin, a natural antifibrotic compound, could remarkably attenuate liver fibrosis in chemically intoxicated rats, but the underlying molecular mechanisms are poorly understood. The present study was aimed at investigating the effects of acupuncture combined with curcumin on platelet-derived growth factor (PDGF) signalling and extracellular matrix (ECM) regulation in the fibrotic liver. A total of 60 Sprague-Dawley male rats were randomly divided into control, model, sham, acupuncture, curcumin and combination treatment groups. During the establishment of fibrosis using carbon tetrachloride (CCl(4)), acupuncture at LR3, LR14, BL18 and ST36 and/or curcumin treatment by mouth were performed simultaneously. After treatment, serum PDGF levels were measured. Protein and mRNA expression of key effectors in PDGF pathway and fibrinolysis in the liver was determined. Acupuncture combined with curcumin potently reduced serum PDGF levels and selectively disrupted the PDGF-βR/extracellular signal-regulated kinase (ERK) cascade. Combination treatment also significantly repressed expression of connective tissue growth factor and upregulated expression of matrix metalloproteinase-9, promoting fibrinolysis in the fibrotic liver. The beneficial effects of acupuncture and its combination with curcumin could be attributed to the disruption of PDGF-βR/ERK pathway and stimulated ECM degradation in the fibrotic liver. Acupuncture treatment significantly enhanced curcumin effects at the molecular level. These findings may provide molecular insights into the potential of acupuncture combined with curcumin for prevention of hepatic fibrosis.

  11. Induction of apoptosis in renal cell carcinoma by reactive oxygen species: involvement of extracellular signal-regulated kinase 1/2, p38delta/gamma, cyclooxygenase-2 down-regulation, and translocation of apoptosis-inducing factor.

    LENUS (Irish Health Repository)

    Ambrose, Monica

    2012-02-03

    Renal cell carcinoma (RCC) is the most common malignancy of the kidney. Unfortunately, RCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of RCCs. We now report that 6-anilino-5,8-quinolinequinone (LY83583), an inhibitor of cyclic GMP production, induced growth arrest and apoptosis of the RCC cell line 786-0. It did not prove deleterious to normal renal epithelial cells, an important aspect of chemotherapy. To address the cellular mechanism(s), we used both genetic and pharmacological approaches. LY83583 induced a time- and dose-dependent increase in RCC apoptosis through dephosphorylation of mitogen-activated protein kinase kinase 1\\/2 and its downstream extracellular signal-regulated kinases (ERK) 1 and -2. In addition, we observed a decrease in Elk-1 phosphorylation and cyclooxygenase-2 (COX-2) down-regulation. We were surprised that we failed to observe an increase in either c-Jun NH(2)-terminal kinase or p38alpha and -beta mitogen-activated protein kinase activation. In contradiction, reintroduction of p38delta by stable transfection or overexpression of p38gamma dominant negative abrogated the apoptotic effect. Cell death was associated with a decrease and increase in Bcl-x(L) and Bax expression, respectively, as well as release of cytochrome c and translocation of apoptosis-inducing factor. These events were associated with an increase in reactive oxygen species formation. The antioxidant N-acetyl l-cysteine, however, opposed LY83583-mediated mitochondrial dysfunction, ERK1\\/2 inactivation, COX-2 down-regulation, and apoptosis. In conclusion, our results suggest that LY83583 may represent a novel therapeutic agent for the treatment of RCC, which remains highly refractory to antineoplastic agents. Our data provide a molecular basis for the anticancer activity of LY83583.

  12. P2Y1 purinoceptor inhibition reduces extracellular signal-regulated protein kinase 1/2 phosphorylation in spinal cord and dorsal root ganglia: implications for cancer-induced bone pain

    Institute of Scientific and Technical Information of China (English)

    Jun Chen; Lina Wang; Yanbing Zhang; Jianping Yang

    2012-01-01

    It remains unclear as to whether P2Y1 purinergic receptor (P2Y1R) and the molecules that act downstream,such as extracellular signal-regulated protein kinase 1/2 (ERK1/2),are involved in the development of cancer-induced bone pain (CIBP) in vivo.Here,we investigated the role of the P2Y1R in the modulation of CIBP-associated nociception in spinal cord and dorsal root ganglia (DRG).A CIBP model was established by inoculating Walker 256 gland carcinoma cells into the tibia of female rats.Tactile ailodynia and spontaneous pain were assessed using von Frey filaments and ambulatory scores.The results showed that both the paw withdrawal latency to tactile allodynia and the ambulatory score to spontaneous pain were significantly different between the CIBP group and the sham group on days 7-9 post-inoculation (P < 0.01).Furthermore,rats in the CIBP group also showed a progressive increase in ambulatory score,which is different from the sham group (P<0.01).Furthermore,P2Y1R mRNA and phosphory lated ERK1/2 (p-ERK1/2) protein expression levels were increased in the spinal dorsal horn and DRG of the CIBP group relative to the sham group.However,intrathecal injection of the P2Y1R antagonist MRS2179 decreased P2Y1R mRNA and p-ERK1/2 protein expression in the spinal dorsal horn and DRG (P<0.01).These results provide evidence that the inhibition of P2Y1R-mediated ERK1/2 phosphorylation in the spinal dorsal horn and DRG can attenuate nociception transmission.

  13. Bis-Retinoid A2E Induces an Increase of Basic Fibroblast Growth Factor via Inhibition of Extracellular Signal-Regulated Kinases 1/2 Pathway in Retinal Pigment Epithelium Cells and Facilitates Phagocytosis

    Science.gov (United States)

    Balmer, Delphine; Bapst-Wicht, Linda; Pyakurel, Aswin; Emery, Martine; Nanchen, Natacha; Bochet, Christian G.; Roduit, Raphael

    2017-01-01

    Age-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for RPE is the best-characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology. Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD. PMID:28298893

  14. Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

    Directory of Open Access Journals (Sweden)

    Fang Huang

    2016-06-01

    Full Text Available Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP. Further, pretreatment with antioxidant N-acetylcysteine (NAC effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125 and ERK1/2 inhibitor (PD98059 effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway.

  15. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    Science.gov (United States)

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  16. Erythropoietin Rescues Primary Rat Cortical Neurons by Altering the Nrf2:Bach1 Ratio: Roles of Extracellular Signal-Regulated Kinase 1/2.

    Science.gov (United States)

    Zhang, Li-Min; Zhang, Dong-Xue; Zhao, Xiao-Chun; Sun, Wenbo

    2017-01-12

    While inhalation anesthetics are indispensable, and generally considered safe and effective, there is growing concern about the selective neurotoxicity of these agents, especially sevoflurane. Erythropoetin (EPO)-induced protection against sevoflurane-induced neuronal death is an effective intervention, but the underlying mechanism is poorly understood. Extracellular signal-related kinases (Erk) 1/2 plays a pivotal role in cell growth and proliferation. Alteration of the nuclear factor erythroid 2-related factor (Nrf2)/BTB-to-CNC homology 1 (Bach1) ratio by Erk1/2 ameliorates the oxidative stress which occurs in human macrophages. Primary cortical neuron cultures exposed to sevoflurane were assessed for Nrf2, Bach1, total Erk1/2, and phosphorylated Erk1/2 with the following: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; propidium iodide uptake; lactate dehydrogenase; malondialdehyde (MDA); superoxide dismutase (SOD); and Western blot. Sevoflurane exposure increased cell death, injury, and MDA (n = 9, P < 0.05), but decreased cell viability and the Nrf2:Bach1 ratio (n = 9, P < 0.05) and down-regulated SOD (n = 9, P < 0.05), while EPO partially rescued the neurotoxicity induced by sevoflurane (n = 9, P < 0.05). Inhibition of Erk1/2 phosphorylation via PD98059 reversed the protective effect of EPO (n = 9, P < 0.05). Thus, protection of EPO markedly attenuated death of neurons exposed to sevoflurane by altering the Nrf2:Bach1 ratio mediated by phosphorylation and activation of Erk1/2.

  17. Dual roles of extracellular signal-regulated kinase (ERK) in quinoline compound BPIQ-induced apoptosis and anti-migration of human non-small cell lung cancer cells.

    Science.gov (United States)

    Fong, Yao; Wu, Chang-Yi; Chang, Kuo-Feng; Chen, Bing-Hung; Chou, Wan-Ju; Tseng, Chih-Hua; Chen, Yen-Chun; Wang, Hui-Min David; Chen, Yeh-Long; Chiu, Chien-Chih

    2017-01-01

    2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11H-indeno[1,2-c]quinoline-11-one (BPIQ), is a synthetic quinoline analog. A previous study showed the anti-cancer potential of BPIQ through modulating mitochondrial-mediated apoptosis. However, the effect of BPIQ on cell migration, an index of cancer metastasis, has not yet been examined. Furthermore, among signal pathways involved in stresses, the members of the mitogen-activated protein kinase (MAPK) family are crucial for regulating the survival and migration of cells. In this study, the aim was to explore further the role of MAPK members, including JNK, p38 and extracellular signal-regulated kinase (ERK) in BPIQ-induced apoptosis and anti-migration of human non-small cell lung cancer (NSCLC) cells. Western Blot assay was performed for detecting the activation of MAPK members in NSCLC H1299 cells following BPIQ administration. Cellular proliferation was determined using a trypan blue exclusion assay. Cellular apoptosis was detected using flow cytometer-based Annexin V/propidium iodide dual staining. Cellular migration was determined using wound-healing assay and Boyden's chamber assay. Zymography assay was performed for examining MMP-2 and -9 activities. The assessment of MAPK inhibition was performed for further validating the role of JNK, p38, and ERK in BPIQ-induced growth inhibition, apoptosis, and migration of NSCLC cells. Western Blot assay showed that BPIQ treatment upregulates the phosphorylated levels of both MAPK proteins JNK and ERK. However, only ERK inhibitor rescues BPIQ-induced growth inhibition of NSCLC H1299 cells. The results of Annexin V assay further confirmed the pro-apoptotic role of ERK in BPIQ-induced cell death of H1299 cells. The results of wound healing and Boyden chamber assays showed that sub-IC50 (sub-lethal) concentrations of BPIQ cause a significant inhibition of migration in H1299 cells accompanied with downregulating the activity of MMP-2 and -9, the

  18. Heat shock protein 90 acts as a molecular chaperone in late-phase acti-vation of extracellular signal-regulated kinase 1/2 stimulated by oxida-tive stress in vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Dai-hua LIU; Hao-yu YUAN; Chun-ya CAO; Zhi-ping GAO; Bing-yang ZHU; Hong-lin HUANG; Duan-fang LIAO

    2007-01-01

    Aim: To investigate whether cytosolic heat shock protein 90 (HSP90) acts as a molecular chaperone on the activated extracellular signal-regulated kinase 1/2 (ERK1/2) and cell proliferation stimulated by reactive oxygen species (ROS) in rat vascular smooth muscle cells (VSMC). Methods: VSMC were exposed to 1 μmol/L LY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in the presence or absence of 5 μmol/L geldanamycin, a specific inhibitor of HSP90. Then the total, soluble, and insoluble proteins of the ceils were collected. HSP90, ERK1/2, and phosphor-ERK 1/2 in the cell lysate were measured by Western blotting. The interaction of HSP90 and phosphor-ERK1/2 was analyzed by immunoprecipi- tation assay, and the nuclear phosphor-ERK1/2 was measured by Western blot- ting and immunofluorescence. Cell proliferation was tested by cell counting and 3-(4,5-dimethylthiazol-2-yl)-3,5-di-phenyltetrazoliumbromide (MTT). Results: The cytosolic HSP90 of VSMC was upregulated by LY83583 in a time-dependent man- ner with the peak at 120 min, which is consistent with the late peak of phosphor- ERK1/2. Immunoprecipitation and Western blotting analyses showed that LY83583 increased the interaction of HSP90 with phosphor-ERK1/2, the phosphor-ERK1/2 level, and the soluble phosphor-ERK1/2 level by 1.8-, 2.5-, and 2.9-fold, respectively. In contrast, the insoluble phosphor-ERK1/2 of VSMC was decreased. Interestingly, LY83583 treatment promoted the nuclear phosphor-ERK1/2 by 7.6-fold as con- finned by Western blotting and immunofluorescence assays. Furthermore, cell counting and the MTT assay showed that LY83583 stimulated VSMC prolifera- tion with the increased expression of HSP90 and levels of soluble and nuclear phosphor-ERK1/2. Pretreatment of geldanamycin antagonized the effect of LY83583. Conclusion: HSP90 could mediate the oxidative stress-stimulated, late- phase activation of ERK1/2 and VSMC proliferation by promoting the ERK1/2 phosphorylation, the

  19. Ras-induced and extracellular signal-regulated kinase 1 and 2 phosphorylation-dependent isomerization of protein tyrosine phosphatase (PTP)-PEST by PIN1 promotes FAK dephosphorylation by PTP-PEST.

    Science.gov (United States)

    Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X; Lu, Zhimin

    2011-11-01

    Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.

  20. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU.

    Science.gov (United States)

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E; Hines, Kevin J; Ragheb, Jonathan; Jog, Neelakshi R; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett A; Cheung, Joseph Y; Kurosaki, Tomohiro; Gill, Donald L; Madesh, Muniswamy

    2015-03-03

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

  1. Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide

    NARCIS (Netherlands)

    S. Feldt (Sandra); W.W. Batenburg (Wendy); I. Mazak (Istvan); U. Maschke (Ulrike); M. Wellner (Maren); H. Kvakan (Heda); R. Dechend (Ralf); A. Fiebeler (Anette); C. Burckle (Celine); A. Contrepas (Aurelie); A.H.J. Danser (Jan); M. Bader (Michael); G. Nguyen (Genevieve); F.C. Luft (Friedrich); D. Müller (Dominik)

    2008-01-01

    textabstractThe recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin p

  2. A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway.

    Science.gov (United States)

    Kim, So Yong; Kim, Tae Jin; Lee, Ki-Young

    2008-06-11

    We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.

  3. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Chun-Yan [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi' an 710032 (China); Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Guo, Xiao-Zhong, E-mail: guoxiaozhong1962@163.com [Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Wang, Hua, E-mail: wanghua@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2011-01-21

    Research highlights: {yields} We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. {yields} Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. {yields} This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  4. Dexmedetomidine Dose-Dependently Attenuates Ropivacaine-Induced Seizures and Negative Emotions Via Inhibiting Phosphorylation of Amygdala Extracellular Signal-Regulated Kinase in Mice.

    Science.gov (United States)

    Zhai, Ming-Zhu; Wu, Huang-Hui; Yin, Jun-Bin; Cui, Yuan-Yuan; Mei, Xiao-Peng; Zhang, Han; Zhu, Xia; Shen, Xue-Feng; Kaye, Alan David; Chen, Guo-Zhong

    2016-05-01

    Ropivacaine (Ropi), one of the newest and safest amino amide local anesthetics, is linked to toxicity, including the potential for seizures, changes in behavior, and even cardiovascular collapse. Dexmedetomidine (Dex), an α2-adrenergic receptor agonist, has been widely used in anesthesia and critical care practice. To date, the underlying mechanisms of the effects of Dex premedication on Ropi-induced toxicity have not been clearly identified. In the current study, we investigated the effects of increasing doses of Dex premedication on 50% convulsive dose (CD50) of Ropi. With increasing doses of intraperitoneal (i.p.) Dex 10 min prior to each i.p. RopiCD50, the latency and duration of seizure activity were recorded. Open-field (OF) and elevated plus maze (EPM) test were used to measure negative behavioral emotions such as depression and anxiety. Immunohistochemistry and Western blot were utilized to investigate phosphorylation-extracellular regulated protein kinases (p-ERK) expression in the basolateral amygdala (BLA) on 2 h and in the central amygdala (CeA) on 24 h after convulsion in mice. The results of our investigation demonstrated that Dex dose-dependently increased RopiCD50, prolonged the latency and shortened the duration of each RopiCD50-induced seizure, improved the negative emotions revealed by both OF and EPM test, and inhibited p-ERK expression in the BLA and the CeA.

  5. Activation of extracellular signal-regulated kinase but not of p38 mitogen-activated protein kinase pathways in lymphocytes requires allosteric activation of SOS.

    Science.gov (United States)

    Jun, Jesse E; Yang, Ming; Chen, Hang; Chakraborty, Arup K; Roose, Jeroen P

    2013-06-01

    Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.

  6. Acupuncture combined with curcumin disrupts platelet-derived growth factor β receptor/extracellular signal-regulated kinase signalling and stimulates extracellular matrix degradation in carbon tetrachloride-induced hepatic fibrosis in rats

    National Research Council Canada - National Science Library

    Zhang, Xiao-Ping; Zhang, Feng; Zhang, Zi-Li; Ma, Jin; Kong, De-Song; Ni, Guang-Xia; Wang, Ai-Yun; Chen, Wen-Xing; Lu, Yin; Zheng, Shi-Zhong

    2012-01-01

    ...) signalling and extracellular matrix (ECM) regulation in the fibrotic liver. A total of 60 Sprague-Dawley male rats were randomly divided into control, model, sham, acupuncture, curcumin and combination treatment groups...

  7. 1-Bromopropane induces macrophage activation via extracellular signal-regulated kinase 1/2 MAPK and NF-κB pathways.

    Science.gov (United States)

    Han, Eun Hee; Hwang, Yong Pil; Lee, Kyung Jin; Jeong, Tae Cheon; Jeong, Hye Gwang

    2008-04-08

    1-Bromopropane (1-BP) has been used in the workplace as an alternative to ozone-depleting solvents. This study examined the effects of 1-BP on the production of nitric oxide (NO) and on proinflammatory cytokines, and analyzed the mechanisms involved in macrophages. 1-BP dose-dependently induced the production of NO and proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, and expression levels of these genes also increased in a dose-dependent manner. The NF-κB sites were identified in the promoter of the iNOS and proinflammatory cytokine genes. Transient transfection and electrophoretic mobility shift assays revealed that NF-κB-mediated the 1-BP-induced increase in the iNOS and proinflammatory cytokine expression levels. Pretreating the macrophages with the NF-κB inhibitor, BAY 11-7082, and the ERK inhibitor, PD98059, inhibited NO production and iNOS expression induced by 1-BP. This demonstrates that 1-BP stimulates macrophage activation via NF-κB transactivation and ERK1/2 MAP kinase phosphorylation. These results suggest that 1-BP has the potential to be inflammatory and that it has previously unrecognized immunomodulating activity.

  8. Ligustrazine attenuates the platelet-derived growth factor-BB-induced proliferation and migration of vascular smooth muscle cells by interrupting extracellular signal-regulated kinase and P38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Yu, Lifei; Huang, Xiaojing; Huang, Kai; Gui, Chun; Huang, Qiaojuan; Wei, Bin

    2015-07-01

    The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) leads to intimal thickening of the aorta and is, therefore, important in the development of arteriosclerosis. As a result, the use of antiproliferative and antimigratory agents for VSMCs offers promise for the treatment of vascular disorders. Although several studies have demonstrated that ligustrazine may be used to treat heart and blood vessel diseases, the detailed mechanism underlying its actions remain to be elucidated. In the present study, the inhibitory effect of ligustrazine on platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation and migration, and the underlying mechanisms were investigated. The findings demonstrated that ligustrazine significantly inhibited PDGF-BB-stimulated VSMC proliferation. VSMCs dedifferentiated into a proliferative phenotype under PDGF-BB stimulation, which was effectively reversed by the administration of ligustrazine. In addition, ligustrazine also downregulated the production of nitric oxide and cyclic guanine monophosphate, induced by PDGF-BB. Additionally, ligustrazine significantly inhibited PDGF-BB-stimulated VSMC migration. Mechanistic investigation indicated that the upregulation of cell cycle-associated proteins and the activation of the extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (MAPK) signaling induced by PDGF-BB was suppressed by the administration of ligustrazine. In conclusion, the present study, demonstrated for the first time, to the best of our knowledge, that ligustrazine downregulated PDGF-BB-induced VSMC proliferation and migration partly, at least, through inhibiting the activation of the ERK and P38 MAPK signaling.

  9. Irciniastatin A induces potent and sustained activation of extracellular signal-regulated kinase and thereby promotes ectodomain shedding of tumor necrosis factor receptor 1 in human lung carcinoma A549 cells.

    Science.gov (United States)

    Quach, Hue Tu; Hirano, Seiya; Fukuhara, Sayuri; Watanabe, Tsubasa; Kanoh, Naoki; Iwabuchi, Yoshiharu; Usui, Takeo; Kataoka, Takao

    2015-01-01

    Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.

  10. Icariin inhibits oxidized low-density lipoprotein-induced proliferation of vascular smooth muscle cells by suppressing activation of extracellular signal-regulated kinase 1/2 and expression of proliferating cell nuclear antigen.

    Science.gov (United States)

    Hu, Yanwu; Liu, Kai; Yan, Mengtong; Zhang, Yang; Wang, Yadi; Ren, Liqun

    2016-03-01

    Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess anti-inflammatory, anti‑oxidant and anti-atherosclerotic activities in vivo and in vitro. The aim of the present study was to investigate the effects of icariin on oxidized low‑density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) and the possible underlying mechanism. VSMCs were cultured and pre‑treated with various concentrations of icariin (0, 10, 20 or 40 µm) prior to stimulation by ox‑LDL (50 µg/ml). Cell proliferation was evaluated by an MTT assay. Flow cytometry was used to study the influence of icariin on the cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 were detected by western blot analysis. The results indicated that icariin significantly inhibited ox‑LDL‑induced proliferation of VSMCs and phosphorylation of ERK1/2. Furthermore, icariin also blocked the ox‑LDL‑induced cell‑cycle progression at G1/S‑interphase and downregulated the expression of PCNA in VSMCs. In conclusion, the present study indicated for the first time that icariin reduced the amount of ox‑LDL‑induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2.

  11. Tanshinone IIA attenuates interleukin-17A-induced systemic sclerosis patient-derived dermal vascular smooth muscle cell activation via inhibition of the extracellular signal-regulated kinase signaling pathway

    Directory of Open Access Journals (Sweden)

    Mengguo Liu

    2015-04-01

    Full Text Available OBJECTIVE: Salvia miltiorrhiza has long been used to treat systemic sclerosis. Tanshinone IIA, one of the phytochemicals derived from the roots of Salvia miltiorrhiza, exhibits multiple biological activities. The present study aimed to investigate whether tanshinone IIA has an effect on the interleukin-17A-induced functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells. METHODS: Systemic sclerosis patient-derived dermal vascular smooth muscle cells were incubated with various dosages of tanshinone IIA in the presence of interleukin-17A or the serum of systemic sclerosis patients. Cell proliferation was assessed using Cell Counting Kit-8. The expression of collagen 1 and 3 in cells was evaluated by immunofluorescence. Cell migration was measured using a transwell assay. The expression of phospho-extracellular signal-regulated kinase was detected by Western blotting. RESULTS: Our data demonstrate that tanshinone IIA exerts an inhibitory effect on interleukin-17A-induced systemic sclerosis patient-derived dermal vascular smooth muscle cell proliferation, collagen synthesis and migration. CONCLUSION: These findings suggest that tanshinone IIA might serve as a promising therapeutic agent for the treatment of systemic sclerosis.

  12. Extracellular matrix mediates epithelial effects on chondrogenesis in vitro.

    Science.gov (United States)

    Solursh, M; Jensen, K L; Zanetti, N C; Linsenmayer, T F; Reiter, R S

    1984-10-01

    It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.

  13. Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2.

    Science.gov (United States)

    Xiao, Yao; Warner, Lisa R; Latham, Michael P; Ahn, Natalie G; Pardi, Arthur

    2015-07-21

    Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for (13)C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of (13)C(1)H3-labeled methyls by comparing distance information obtained from three-dimensional methyl-methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl-methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed "NOE-methyl cluster" onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV (13)C(1)H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.

  14. Curcumin exerts antinociceptive effects by inhibiting the activation of astrocytes in spinal dorsal horn and the intracellular extracellular signal-regulated kinase signaling pathway in rat model of chronic constriction injury

    Institute of Scientific and Technical Information of China (English)

    JI Feng-tao; LIANG Jiang-jun; LIU Ling; CAO Ming-hui; LI Feng

    2013-01-01

    Background Activation of glial cells and the extracellular signal-regulated kinase (ERK) signaling pathway play an important role in the development and maintenance of neuropathic pain.Curcumin can alleviate the symptom of inflammatory pain by inhibiting the production and release of interleukin and tumor necrosis factor.However,whether curcumin affects neuropathic pain induced by nerve injury and the possible mechanism involved are still unknown.This study investigated the effects of tolerable doses of curcumin on the activation of astrocytes and ERK signaling in the spinal dorsal horn in rat model of neuropathic pain.Methods Adult male Sprague-Dawley rats were randomly divided into three groups:a control (sham operated) group,and chronic constriction injury groups (to induce neuropathic pain) that were either untreated or treated with curcumin.Thermal and mechanical hyperalgesia thresholds were measured.The distribution and morphological changes of astrocytes were observed by immunofluorescence.Western blotting was used to detect changes in the expression of glial flbrillary acid protein (GFAP) and phosphorylated ERK.Results Injured rats showed obvious mechanical allodynia and thermal hyperalgesia.The number of GFAP-positive astrocytes,and the fluorescence intensity of GFAP were significantly increased in the spinal dorsal horn of injured compared with control rats.The soma of astrocytes also appeared hypertrophied in injured animals.Expression of GFAP and phosphorylated ERK was also significantly increased in the spinal dorsal hom of injured compared with control rats.Curcumin reduced the injury-induced thermal and mechanical hyperalgesia,the increase in the fluorescence intensity of GFAP and the hypertrophy of astrocytic soma,activation of GFAP and phosphorylation of ERK in the spinal dorsal horn.Conclusions Curcumin can markedly alleviate nerve injury-induced neuropathic pain in rats.The analgesic effect of curcumin may be attributed to its inhibition of

  15. Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells.

    Science.gov (United States)

    Herraiz, Cecilia; Journé, Fabrice; Ghanem, Ghanem; Jiménez-Cervantes, Celia; García-Borrón, José C

    2012-12-01

    Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.

  16. Pharmacological Activation Gi/o Protein Increases Glial Cell Line-Derived Neurotrophic Factor Production through Fibroblast Growth Factor Receptor and Extracellular Signal-Regulated Kinase Pathway in Primary Cultured Rat Cortical Astrocytes.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Matsumoto, Chie; Azuma, Honami; Taki, Sayaka; Takebayashi, Minoru; Nakata, Yoshihiro; Morioka, Norimitsu

    2017-01-01

    A significant reduction of glial cell line-derived neurotrophic factor (GDNF) has been identified in the pathophysiology of neurodegenerative and neuropsychiatric disorders. Thus, clarification of the mechanism of GDNF production, and modulating brain GDNF levels could be a novel therapeutic approach. A previous study demonstrated that antidepressant amitriptyline-induced GDNF production was significantly inhibited by pertussis toxin (PTX), a Gi/o protein inhibitor in astrocytes, the main source of GDNF in the brain. However, it is not known whether direct activation of Gi/o protein might induce GDNF expression, and what mechanisms might be involved after Gi/o protein activation. The current study investigated Gi/o protein-initiated GDNF production in rat cortical astrocytes using activators that directly activate Gi/o protein, mastoparan and compound48/80. Treatment of astrocytes with either mastoparan or compound48/80 increased GDNF mRNA expression at 3 and 6 h, and GDNF protein release at 24 h. Treatment of astrocyte with either mastoparan or compound48/80 increased brain-derived neurotrophic factor (BDNF) mRNA expression as well as GDNF. Mastoparan and compound48/80-induced GDNF mRNA expression were significantly inhibited by not only PTX, but also fibroblast growth factor receptor (FGFR) inhibitors, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. In fact, both FGFR substrate2α (FRS2α) and ERK phosphorylation were increased by treatment with either mastoparan or compound48/80, and these were significantly blocked by PTX. Thus, direct, receptor-independent Gi/o protein activation increases GDNF production through FGFR/ERK signaling pathway. The current results indicate a critical role of Gi/o signaling in the regulation of GDNF expression in astrocytes.

  17. Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

    Science.gov (United States)

    Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

    2014-03-01

    Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories.

  18. Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice.

    Science.gov (United States)

    Moriguchi, Shigeki; Yamamoto, Yui; Ikuno, Tatsuya; Fukunaga, Kohji

    2011-06-01

    Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  19. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

    Directory of Open Access Journals (Sweden)

    Sanja Perkovic

    2012-09-01

    Full Text Available Aggressive natural killer-cell leukaemia (ANKL is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV and P-glycoprotein (P-gp expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2 MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  20. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells.

    Science.gov (United States)

    Liu, Xiang; Du, Liying; Feng, Renqing

    2013-07-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells. Here, we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Western blot analysis demonstrated the down-regulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2. Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3β). Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKT pathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression. The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity, whereas the p27 Kip1 expression was increased. In addition, knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2, AKT, and GSK3β. After c-Src depletion by siRNAs, we observed significant down-regulation of cyclin D1 and cyclin E, and up-regulation of p27 Kip1. These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  1. Depletion of Serotonin and Selective Inhibition of 2B Receptor Suppressed Tumor Angiogenesis by Inhibiting Endothelial Nitric Oxide Synthase and Extracellular Signal-Regulated Kinase 1/2 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Masanori Asada

    2009-04-01

    Full Text Available The effects of serotonin (5-HT on tumor growth are inconsistent. We investigated whether a decreased level of 5-HT affected tumor growth using 5-HT transporter knockout (5-HTT-/- mice, which showed 5-HT depletion. When cancer cells were injected subcutaneously into both 5-HTT-/- and 5-HTT+/+ mice, the tumor growth was markedly attenuated in 5-HTT-/- mice. Serotonin levels in the blood, forebrain, and tumors of 5-HTT-/- mice bearing tumors were significantly smaller than those of their 5-HTT+/+ littermates. However, 5-HT did not increase cancer cells' proliferation in vitro. When we applied 5-HTT inhibitors to the wild mice bearing tumors, they did not inhibit tumor growth. The endothelial nitric oxide synthase (eNOS expressions in tumors were reduced in 5-HTT-/- mice compared with 5-HTT+/+ mice. Stimulations with 5-HT (1–50 µM induced eNOS expressions in human umbilical vein endothelial cell (HUVEC in a concentration-dependent manner. When we measured activations of multiple signaling pathways by using a high-throughput phosphospecific antibodies platform, 5-HT stimulated the extracellular signal-regulated kinase 1/2 (ERK1/2 in HUVEC. Moreover, we found that the physiological level of 5-HT induced phosphorylation of both ERK1/2 and eNOS in HUVEC. Human umbilical vein endothelial cell expressed both 5-HT2B and 5-HT2C receptors. SB204741, a specific 5-HT2B receptor inhibitor, blocked 5-HT-induced ERK1/2 and eNOS phosphorylations, whereas RS102221, a specific 5-HT2C receptor inhibitor, did not in HUVEC. SB204741 reduced microvessel density in tumors and inhibited the proliferation of HUVEC in vitro. These results suggest that regulation of 5-HT and 5-HT receptors, especially the 5-HT2B receptor, may serve as a therapeutic strategy in cancer therapy.

  2. The Role of Matrine and Mitogen-Ativated Protein Kinase/Extracellular Signal-Regulated Kinase Signal Transduction in the Inhibition of the Proliferation and Migration of Human Umbilical Veins Endothelial Cells Induced by Lung Cancer cells

    Directory of Open Access Journals (Sweden)

    Ming BAI

    2009-07-01

    Full Text Available Background and objective Matrine, one of the major alkaloid components of the traditional Chinese medicine Sophora roots, has a wide range of pharmacological effects including anti-inflammatory activities, growth inhibition and induction of cell differentiation and apoptosis. Motigen-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK has found to be a crucial signaling pathway in endothelial cells. The aim of this study is to investigate the role of Matrine and MAPK/ERK signal transduction in the inhibition of the proliferation and migration of human umbilical veins endothelial cells (HUVECs induced by lung cancer cells. Methods HUVECs were cultured with A549CM. Mat or PD98059 (i.e PD, specific inhibitor of MAPK/ERK, was added into the A549CM. The proliferation of the HUVECs was measured by cell counting. The migration of the HUVECs was observed by wound healing assay. The expression levels of ERK and p-ERK protein were detected by Western Blot analysis. Results On 24 hours after intervention, the A549CM significantly stimulated the proliferation, migration and expression of p-ERK of HUVECs. Compared with the A549CM group, Mat significantly inhibited the proliferation, migration and p-ERK expression of HUVECs induced by A549CM. While PD only decreased the proliferation and p-ERK expression of HUVECs induced by A549CM. PD had no effect in the migration of HUVECs. Conclusion The results demonstrated that Mat and PD98059 can effectively decrease proliferation and expression of p-ERK of HUVECs induced by A549CM. Furthermore Mat can also inhibit migration of HUVECs induced by A549CM that did not changed by PD98059. These data implied that suppressing MAPK/ERK signal transduction may play the crucial role in resisting lung cacinoma angiogenesis with Mat.

  3. DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

    Science.gov (United States)

    Yu, Seon-Mi; Kim, Song-Ja

    2015-10-01

    The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells.

  4. Effect of stimulus pre-exposure on inhibitory avoidance retrieval-associated changes in the phosphorylated form of the extracellular signal-regulated kinase-1 and -2 (pERK1/2).

    Science.gov (United States)

    Wang, Chia-Chuan; Chai, Sin-Chee; Holahan, Matthew R

    2010-01-01

    One goal of the present study was to determine how pre-exposure to a set of contextual cues affected subsequent reinforced inhibitory avoidance task performance using those cues (latent inhibition model). In addition, immunohistochemical assessment of the phosphorylated (activated) form of the extracellular signal-regulated kinase-1 and -2 (pERK1/2) was examined. Adult, male Long Evans rats were randomly assigned into either pre-exposure (PE) or different pre-exposure (DPE) groups. All rats received 3days of contextual pre-exposure (same or different context as that used for reinforced training) and were trained, 24h later, on an inhibitory avoidance task (with or without shock). Rats were euthanized 24h after training; half with a retention test and half without. Behaviorally, the PE group showed reduced latencies to enter the dark/shock compartment during the retention test compared to the DPE group showing the latent inhibition phenomenon. Compared to the shocked and tested DPE group, the shocked and tested PE group showed fewer pERK1/2-ir neurons in the secondary motor cortex, the anterior cingulate, the pre- and infra-limbic cortices, and the central nucleus of the amygdala. These regions showed similar numbers of pERK1/2-labeled neurons when comparing the shocked and tested PE group with the nonshocked and tested PE group. This suggests the possibility that brain regions showing decreased pERK1/2 levels in association with attenuated inhibitory avoidance performance may be involved in different aspects of the memory retrieval process.

  5. c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Xiang Liu; Liying Du; Renqing Feng

    2013-01-01

    We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells.Here,we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2).Western blot analysis demonstrated the downregulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2.Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2),protein kinase B (AKT),and glycogen synthase kinase 3 beta (GSK3β).Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKTpathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression.The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity,whereas the p27 Kip1 expression was increased.In addition,knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2,AKT,and GSK3β.After c-Src depletion by siRNAs,we observed significant down-regulation of cyclin D1 and cyclin E,and up-regulation of p27 Kip1.These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3β and ERK1/2 pathways.

  6. Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes.

    Science.gov (United States)

    Cailotto, Frederic; Bianchi, Arnaud; Sebillaud, Sylvie; Venkatesan, Narayanan; Moulin, David; Jouzeau, Jean-Yves; Netter, Patrick

    2007-01-01

    ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an

  7. Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning.

    Science.gov (United States)

    Lin, P-Y; Wang, S-P; Tai, M-Y; Tsai, Y-F

    2010-11-24

    Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is

  8. Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

    Science.gov (United States)

    Tran, Doan Duy Hai; Koch, Alexandra; Saran, Shashank; Armbrecht, Marcel; Ewald, Florian; Koch, Martina; Wahlicht, Tom; Wirth, Dagmar; Braun, Armin; Nashan, Björn; Gaestel, Matthias; Tamura, Teruko

    2016-05-01

    Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.

  9. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Yihui [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Xue, Huaming [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Francis, Wendy [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Davies, Andrew P. [Department of Orthopaedics and Trauma, Moriston Hospital, Swansea (United Kingdom); Pallister, Ian; Kanamarlapudi, Venkateswarlu [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Xia, Zhidao, E-mail: zhidao.xia@gmail.com [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom)

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  10. Extracellular vesicles as mediators of neuron-glia communication

    Directory of Open Access Journals (Sweden)

    Carsten eFrühbeis

    2013-10-01

    Full Text Available In the nervous system, glia cells maintain homeostasis, synthesize myelin, provide metabolic support, and participate in immune defense. The communication between glia and neurons is essential to synchronize these diverse functions with brain activity. Evidence is accumulating that secreted extracellular vesicles (EVs, such as exosomes and shedding microvesicles, are key players in intercellular signaling. Among others, the cells of the nervous system secrete EVs, which carry protein and RNA cargo from one cell to another. After delivery, the cargo has the ability to modify the target cell phenotype. Here, we review the recent advances in understanding the role of EV secretion by astrocytes, microglia, and oligodendrocytes in the central nervous system. Current work has demonstrated that oligodendrocytes transfer exosomes to neurons as a result of neurotransmitter signaling suggesting that these vesicles may mediate glial support of neurons.

  11. Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

    Science.gov (United States)

    Meng, Hong-Zheng; Zhang, Wei-Lin; Liu, Fei; Yang, Mao-Wei

    2015-11-20

    The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

  12. Cellular Traction Stresses Mediate Extracellular Matrix Degradation by Invadopodia

    Science.gov (United States)

    Jerrell, Rachel J.; Parekh, Aron

    2014-01-01

    During tumorigenesis, matrix rigidity can drive oncogenic transformation via altered cellular proliferation and migration. Cells sense extracellular matrix (ECM) mechanical properties with intracellular tensile forces generated by actomyosin contractility. These contractile forces are transmitted to the matrix surface as traction stresses which mediate mechanical interactions with the ECM. Matrix rigidity has been shown to increase proteolytic ECM degradation by cytoskeletal structures known as invadopodia that are critical for cancer progression suggesting that cellular contractility promotes invasive behavior. However, both increases and decreases in traction stresses have been associated with metastatic behavior. Therefore, the role of cellular contractility in invasive migration leading to metastasis is unclear. To determine the relationship between cellular traction stresses and invadopodia activity, we characterized the invasive and contractile properties of an aggressive carcinoma cell line utilizing polyacrylamide gels of different rigidities. We found that ECM degradation and traction stresses were linear functions of matrix rigidity. Using calyculin A to augment myosin contractility, we also found that traction stresses were strongly predictive of ECM degradation. Overall, our data suggest that cellular force generation may play an important part in invasion and metastasis by mediating invadopodia activity in response to the mechanical properties of the tumor microenvironment. PMID:24412623

  13. DMPD: Fragments of extracellular matrix as mediators of inflammation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18243041 Fragments of extracellular matrix as mediators of inflammation. Adair-Kirk...l) Show Fragments of extracellular matrix as mediators of inflammation. PubmedID 18243041 Title Fragments of extrace... TL, Senior RM. Int J Biochem Cell Biol. 2008;40(6-7):1101-10. Epub 2007 Dec 24. (.png) (.svg) (.html) (.csm

  14. The extracellular signal-regulated kinase was promoted by Pyrroloquinoline Quinine in cultured Schwann cells%MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用

    Institute of Scientific and Technical Information of China (English)

    贺斌; 刘世清; 李皓桓

    2010-01-01

    目的 探讨MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖过程中的作用. 方法 体外培养雪旺细胞,S-100免疫荧光鉴定;Western blot检测MEK下游因子ERK1/2磷酸化激活形式(p-ERK1/2)的表达;MEK抑制剂(PD98059)阻断该通路后检测p-ERK1/2的表达;MTT法检测经PD98059阻断MEK通路后雪旺细胞的增殖情况. 结果 吡咯喹啉醌可激活雪旺细胞内MEK/ERK信号通路,在加入吡咯喹啉醌1 h后p-ERK1/2表达最高;吡咯喹啉醌在1~500 nmol/L范围内可使p-ERK1/2表达增加,1 000 nmol/L时与对照组比较差异无统计学意义,10 000 nmol/L时则表现为抑制作用(P<0.05);经PD98059阻断MEK通路后p-ERK1/2的上调效应消失(P<0.05).而且加入PD98059阻断MEK通路后吡咯喹啉醌对雪旺细胞的促增殖效果减弱. 结论 吡咯喹啉醌可激活雪旺细胞MEK/ERK信号通路,且该通路在吡咯喹啉醌促雪旺细胞增殖过程中发挥作用.%Objective To investigate the effect of mitogen-activated protein kinase(MEK)kinase cascade,extracellular signal-regulated kinase(ERK1/2)signal pathway on Schwann cells proliferation promoted by Pyrroloquinoline Quinine(PQQ)and its molecular mechanisms. Methods Schwann cells were cultured and purified in vitro.The purity was identified by S-100.Different time and concentration of PQQ was added into culture medium.The expression of ERK1/2 and phosphorylated-ERK1/2 was detected by western blot.The expression of p-ERK1/2 after blocking of MEK signal pathway by specific inhibitor PD98059 was detected by western blot. Results Morphological change was observed in PQQ treated Schwann cells.1 ~ 500 nmol/L PQQ could up-regulate the expression of p-ERK1/2,and 1 000 nmol/Lhad no effects,while 10 000 nmol/L exhibited inhibitory effect(P<0.05).p-ERKI/2 increased to peak 1 h after PQQ added,and this up-regulation of p-ERKI/2 was inhibited by PD98059(P<0.05). Conclusions PQQ could affect morphology of Schwann cells and activation of ERKI/2

  15. Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

    Science.gov (United States)

    Möllerherm, Helene; Neumann, Ariane; Schilcher, Katrin; Blodkamp, Stefanie; Zeitouni, Nathalie E; Dersch, Petra; Lüthje, Petra; Naim, Hassan Y; Zinkernagel, Annelies S; von Köckritz-Blickwede, Maren

    2015-12-01

    Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

  16. Sevoflurane Post-conditioning Protects Primary Rat Cortical Neurons Against Oxygen-Glucose Deprivation/Resuscitation: Roles of Extracellular Signal-Regulated Kinase 1/2 and Bid, Bim, Puma.

    Science.gov (United States)

    Zhang, Limin; Zhao, Xiaochun; Jiang, Xiaojing

    2015-08-01

    Temporal post-conditioning to induce neuroprotection against brain ischemia-reperfusion injury insult is considered to be an effective intervention, but the exact mechanisms of sevoflurane post-conditioning are poorly understood. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in the cell growth and proliferation. The essential axis of activator Bid, Bim, Puma (BH3s) and BAX, BAK in activating the mitochondrial death program might offer common ground for cell death signal. We hypothesized that, sevoflurane post-conditioning might inhibit the expression of Bid, Bim and Puma and is activated by phosphor-Erk1/2 to reduce neuronal death. To test this hypothesis, we exposed primary cultured cortical neurons to oxygen-glucose deprivation for 1 h and resuscitation for 24 h (OGD/R). The assays of MTT, propidium iodide uptake, JC-1 fluorescence and western blot demonstrated that OGD/R exposure reduced cell viability, increased cell death, decreased mitochondrial membrane potential and the expressions of Bid, Bim, and Puma. Inhibition of Erk1/2 phosphorylation could partially attenuate 2 % of sevoflurane post-conditioning mediated increase in neuronal viability and mitochondrial membrane potential, and also a decrease in cell death and expression of Bid, Bim and Puma after OGD/R treatment. The results demonstrated that, the protection of sevoflurane post-conditioning markedly reducing death of cortical neurons exposed to OGD/R could be correlated with down-regulation of Bid, Bim and Puma expression mediated by phosphorylation/activation of Erk1/2.

  17. Role of leptin signaling in hemato-vascular development and niche function: Leptin receptor-mediated signaling regulates LT-HSC homeostasis in vivo

    Science.gov (United States)

    Homeostatic functioning of the cardiovascular and hematopoietic systems is known to be interdependent and strongly influenced by the microenvironment in which hemato-vascular cells develop and reside. The role of nutrition and metabolism as regulable and dynamic extracellular cues however, remains a...

  18. Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2009-01-01

    Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

  19. Extracellular Electron Uptake: Among Autotrophs and Mediated by Surfaces

    DEFF Research Database (Denmark)

    Tremblay, Pier-Luc; Angenent, Largus T.; Zhang, Tian

    2017-01-01

    Autotrophic microbes can acquire electrons from solid donors such as steel, other microbial cells, or electrodes. Based on this feature, bioprocesses are being developed for the microbial electrosynthesis (MES) of useful products from the greenhouse gas CO2. Extracellular electron-transfer mechan......Autotrophic microbes can acquire electrons from solid donors such as steel, other microbial cells, or electrodes. Based on this feature, bioprocesses are being developed for the microbial electrosynthesis (MES) of useful products from the greenhouse gas CO2. Extracellular electron...

  20. Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

    DEFF Research Database (Denmark)

    Goettsch, Claudia; Hutscheson, JD; Aikawa, M

    2016-01-01

    obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin...

  1. Pseudomonas deceptionensis DC5-mediated synthesis of extracellular silver nanoparticles.

    Science.gov (United States)

    Jo, Jae H; Singh, Priyanka; Kim, Yeon J; Wang, Chao; Mathiyalagan, Ramya; Jin, Chi-Gyu; Yang, Deok C

    2016-09-01

    The biological synthesis of metal nanoparticles is of great interest in the field of nanotechnology. The present work highlights the extracellular biological synthesis of silver nanoparticles using Pseudomonas deceptionensis DC5. The particles were synthesized in the culture supernatant within 48 h of incubation. Extracellular synthesis of silver nanoparticles in the culture supernatant was confirmed by ultraviolet-visible spectroscopy, which showed the absorption peak at 428 nm, and also under field emission transmission electron microscopy which displayed the spherical shape. In addition, the particles were characterized by X-ray diffraction spectroscopy, which corresponds to the crystalline nature of nanoparticles, and energy-dispersive X-ray analysis which exhibited the intense peak at 3 keV, resembling the silver nanoparticles. Further, the synthesized nanoparticles were examined by elemental mapping which displayed the dominance of the silver element in the synthesized product, and dynamic light scattering which showed the distribution of silver nanoparticles with respect to intensity, volume, and number of particles. Moreover, the silver nanoparticles have been found to be quite active in antimicrobial activity and biofilm inhibition activity against pathogenic microorganisms. Thus, the present work emphasized the prospect of using the P. deceptionensis DC5 to achieve the extracellular synthesis of silver nanoparticles in a facile and environmental manner.

  2. Extracellular signal regulated kinase and SMAD signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR(mRen2)27

    NARCIS (Netherlands)

    de Boer, RA; Pokharel, S; Flesch, M; van Kampen, DA; Suurmeijer, AJH; Boomsma, F; van Gilst, WH; van Veldhuisen, DJ; Pinto, YM

    2004-01-01

    Angiotensin (Ang) II is a key player in left ventricular (LV) remodeling and cardiac fibrosis. Its effects are thought to be transferred at least in part by mitogen-activated protein kinases (MAPK), transforming growth factor (TGF) beta(1), and the Smad pathway. In this study we sought to elucidate

  3. Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death

    DEFF Research Database (Denmark)

    Hollmann, C Annette; Owens, Trevor; Nalbantoglu, Josephine;

    2006-01-01

    CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models...... a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines...... and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40....

  4. Extracellular signal-regulated kinase (ERK) signaling in the ventral tegmental area mediates cocaine-induced synaptic plasticity and rewarding effects

    OpenAIRE

    Pan, Bin; Zhong, Peng; Sun, Dalong; Liu, Qing-song

    2011-01-01

    Drugs of abuse such as cocaine induce long-term synaptic plasticity in the reward circuitry, which underlies the formation of drug-associated memories and addictive behavior. We reported previously that repeated cocaine exposure in vivo facilitates long-term potentiation in dopamine neurons of the ventral tegmental area (VTA) by reducing the strength of GABAergic inhibition and that endocannabinoid (eCB)-dependent long-term depression at inhibitory synapses (I-LTD) constitutes a mechanism for...

  5. The bHLH factors Dpn and members of the E(spl complex mediate the function of Notch signalling regulating cell proliferation during wing disc development

    Directory of Open Access Journals (Sweden)

    Beatriz P. San Juan

    2012-05-01

    The Notch signalling pathway plays an essential role in the intricate control of cell proliferation and pattern formation in many organs during animal development. In addition, mutations in most members of this pathway are well characterized and frequently lead to tumour formation. The Drosophila imaginal wing discs have provided a suitable model system for the genetic and molecular analysis of the different pathway functions. During disc development, Notch signalling at the presumptive wing margin is necessary for the restricted activation of genes required for pattern formation control and disc proliferation. Interestingly, in different cellular contexts within the wing disc, Notch can either promote cell proliferation or can block the G1-S transition by negatively regulating the expression of dmyc and bantam micro RNA. The target genes of Notch signalling that are required for these functions have not been identified. Here, we show that the Hes vertebrate homolog, deadpan (dpn, and the Enhancer-of-split complex (E(splC genes act redundantly and cooperatively to mediate the Notch signalling function regulating cell proliferation during wing disc development.

  6. TNFα signaling regulates cystic epithelial cell proliferation through Akt/mTOR and ERK/MAPK/Cdk2 mediated Id2 signaling.

    Directory of Open Access Journals (Sweden)

    Julie X Zhou

    Full Text Available Tumor necrosis factor alpha (TNFα is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD. However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand, a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.

  7. BRAF及其细胞外信号调节激酶1/2信号通路蛋白在甲状腺乳头状癌中的表达及意义%Expression of BRAF and its extracellular signal-regulated kinase 1/2 signal pathway in papillary thyroid cancer

    Institute of Scientific and Technical Information of China (English)

    易文君; 钟德玝; 邹琼燕

    2012-01-01

    目的:研究BRAF基因及其丝裂原活化蛋白/胞外信号调节激酶的激酶(mitogen-activated protein/extracellular signal-regulated kinase kinase,MEK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号通路与甲状腺乳头状癌发生的相关性及部分作用机制.方法:收集73例散发的甲状腺乳头状癌患者及16例同期甲状腺瘤患者(对照组)的临床资料及标本.采用免疫组织化学和免疫印迹法检测两组标本组织中的大鼠肉瘤蛋白(rat sarcoma,RAS),BRAF,MEK1/2和ERK1/2表达状况.结果:甲状腺乳头状癌组RAS,BRAF,pMEK1/2和pERK1/2表达水平明显高于甲状腺瘤组(P<0.05或P<0.01);甲状腺乳头状癌患者RAS,BRAF,pMEK1/2和pERK1/2表达与肿块大小、淋巴结转移以及临床分期相关(P<0.05或P<0.01).结论:RAS,BRAF,pMEK1/2及pERK1/2可能与甲状腺乳头状癌发生及其淋巴结转移、临床分期相关;BRAF可能通过激活MEK/ERK信号通路而发挥其生物作用.%Objective: To determine the association between activity of BRAF and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) / extracellular signal-regulated kinase (ERK)signal pathway in papillary thyroid cancer and its mechanism.Methods: We collected the clinical data and blood samples from 73 cases of papillary thyroid cancer and another 16 cases of benign thyroid gland tumor, and detected the expression of rat sarcoma (RAS), BRAF, MEK1/2, and ERK1/2 in all tumor specimens and benign thyroid tissues with immunohistochemistry and Western blot. Results: The expression of RAS, BRAF, pMEKl/2, and pERKl/2 protein in papillary thyroid cancer tissues was higher than those in the benign thyroid tissues(P<0.05 or P<0.0l). The expression of RAS, BRAF, MEK1/2, and ERKl/2 was associated with the tumor size, the lymph node metastasis, and the clinical stage of papillary thyroid cancer(P<0.05 orP<0.0l).Conclusion: The expression of RAS, BRAF, pMEKl/2, and pERKl/2 is

  8. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells.

    Science.gov (United States)

    Cho, Sung-Yun; Lee, Hyo-Jeong; Lee, Hyo-Jung; Jung, Deok-Beom; Kim, Hyunseok; Sohn, Eun Jung; Kim, Bonglee; Jung, Ji Hoon; Kwon, Byoung-Mog; Kim, Sung-Hoon

    2013-01-01

    Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPK α blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPK α in hypoxic SW620 cells, implying cross-talk between ERK and AMPK α . Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1 α and Akt/mTOR and the activation of AMPK α and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPK α in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPK α and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

  9. 低功率激光照射诱导的细胞外调节蛋白激酶的激活促进一氧化氮的产生%Low-power Laser Irradiation Stimulates Nitric Oxide Production through Activating Extracellular Signal-regulated Protein Kinase

    Institute of Scientific and Technical Information of China (English)

    冯杰; 吴胜男

    2012-01-01

    Low-power laser irradiation ( LPLI) , as a non-damage physical therapy, acclerrates cell survival, angiogen-esis and wound healing in which nitric oxide (NO) acts as an important contributor. NO promotes the tendency of inflammation cell, increases the synthesis and precipitation of collagen, and stimulates cell proliferation and angiogenesis. In this study, we found that LPLI boosted the production of NO, which was inhibited by blocking ERK ( Extracellular signal-regulated protein kinase, ERK) activation, indicating LPLI promoted NO generation through activating ERK. The research will supply potential strategy for the clinical application of LPLI in the treatment of wound healing.%低功率激光(632.8 nm)照射(Low-power laser irradiation,LPLI)生物组织作为一种无损伤的物理疗法,可以加速细胞生长、血管再生及伤口愈合等过程.一氧化氮( Nitric oxide,NO)是伤口愈合的关键因素之一,其促进炎性细胞的趋化,增强胶原的合成和沉积,刺激细胞增殖和新生血管生成.我们研究发现LPLI可以促进NO的产生,并且抑制细胞外调节蛋白激酶(Extracellular signal-regulated protein kinases,ERK)的活性阻碍了NO的产生,证明LPLI通过活化ERK调控NO的生成.这一研究将为低功率激光照射加速伤口愈合在临床上的应用奠定基础.

  10. MicroRNA-Mediated Down-Regulation of Apoptosis Signal-Regulating Kinase 1 (ASK1 Attenuates the Apoptosis of Human Mesenchymal Stem Cells (MSCs Transplanted into Infarcted Heart

    Directory of Open Access Journals (Sweden)

    Chang Youn Lee

    2016-10-01

    Full Text Available Stem cell therapy using adult stem cells, such as mesenchymal stem cells (MSCs has produced some promising results in treating the damaged heart. However, the low survival rate of MSCs after transplantation is still one of the crucial factors that limit the therapeutic effect of stem cells. In the damaged heart, oxidative stress due to reactive oxygen species (ROS production can cause the death of transplanted MSCs. Apoptosis signal-regulating kinase 1 (ASK1 has been implicated in the development of oxidative stress-related pathologic conditions. Thus, we hypothesized that down-regulation of ASK1 in human MSCs (hMSCs might attenuate the post-transplantation death of MSCs. To test this hypothesis, we screened microRNAs (miRNAs based on a miRNA-target prediction database and empirical data and investigated the anti-apoptotic effect of selected miRNAs on human adipose-derived stem cells (hASCs and on rat myocardial infarction (MI models. Our data indicated that miRNA-301a most significantly suppressed ASK1 expression in hASCs. Apoptosis-related genes were significantly down-regulated in miRNA-301a-enriched hASCs exposed to hypoxic conditions. Taken together, these data show that miRNA-mediated down-regulation of ASK1 protects MSCs during post-transplantation, leading to an increase in the efficacy of MSC-based cell therapy.

  11. The extracellular EXO protein mediates cell expansion in Arabidopsis leaves.

    Science.gov (United States)

    Schröder, Florian; Lisso, Janina; Lange, Peggy; Müssig, Carsten

    2009-02-13

    The EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described. The exo loss of function mutant showed diminished leaf and root growth and reduced biomass production. Light and scanning electron microscopy analyses revealed that impaired leaf growth is due to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were smaller in comparison to the wild-type. The exo mutant showed reduced brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were significantly more sensitive to the inhibitory effect of synthetic brassinolide. Apart from reduced growth, exo did not show severe morphological abnormalities. Gene expression analyses of leaf material identified genes that showed robust EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and genes involved in primary and secondary metabolism showed weaker expression in exo than in wild-type plants. However, the vast majority of BR-regulated genes were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to the apoplast. The EXO gene is essential for cell expansion in leaves. Gene expression patterns and growth assays suggest that EXO mediates BR-induced leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the shoot. EXO presumably is involved in a signalling process which coordinates BR-responses with environmental or developmental signals. The hypersensitivity of exo roots to BR suggests that EXO plays a diverse role in the control of BR responses in the root.

  12. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia.

    Science.gov (United States)

    Abraham, E; Arcaroli, J; Shenkar, R

    2001-01-01

    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

  13. Extracellular Protein Interactions Mediated by the Neural Cell Adhesion Molecule, NCAM: Heterophilic Interactions Between NCAM and Cell Adhesion Molecules, Extracellular Matrix Proteins, and Viruses

    DEFF Research Database (Denmark)

    Nielsen, Janne; Kulahin, Nikolaj; Walmod, Peter

    2008-01-01

    Cell adhesion molecules (CAMs) mediate cell-to-cell interactions and interactions between cells and the extracellular matrix (ECM). The neural cell adhesion molecule (NCAM), a prototypic member of the immunoglobulin (Ig) superfamily of CAMs, mediates adhesion through homophilic and heterophilic i...

  14. NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV Nonstructural Protein 5A (NS5A via MEK/Extracellular Signal Regulated Kinase (ERK Pathway

    Directory of Open Access Journals (Sweden)

    Xuesong Gao

    2013-05-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein 5A (NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

  15. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

    Science.gov (United States)

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-11-23

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

  16. Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma.

    Science.gov (United States)

    Zhou, Bingying; Ritt, Daniel A; Morrison, Deborah K; Der, Channing J; Cox, Adrienne D

    2016-08-19

    The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.

  17. Extracellular acidosis impairs P2Y receptor-mediated Ca(2+) signalling and migration of microglia.

    Science.gov (United States)

    Langfelder, Antonia; Okonji, Emeka; Deca, Diana; Wei, Wei-Chun; Glitsch, Maike D

    2015-04-01

    Microglia are the resident macrophage and immune cell of the brain and are critically involved in combating disease and assaults on the brain. Virtually all brain pathologies are accompanied by acidosis of the interstitial fluid, meaning that microglia are exposed to an acidic environment. However, little is known about how extracellular acidosis impacts on microglial function. The activity of microglia is tightly controlled by 'on' and 'off' signals, the presence or absence of which results in generation of distinct phenotypes in microglia. Activation of G protein coupled purinergic (P2Y) receptors triggers a number of distinct behaviours in microglia, including activation, migration, and phagocytosis. Using pharmacological tools and fluorescence imaging of the murine cerebellar microglia cell line C8B4, we show that extracellular acidosis interferes with P2Y receptor-mediated Ca(2+) signalling in these cells. Distinct P2Y receptors give rise to signature intracellular Ca(2+) signals, and Ca(2+) release from stores and Ca(2+) influx are differentially affected by acidotic conditions: Ca(2+) release is virtually unaffected, whereas Ca(2+) influx, mediated at least in part by store-operated Ca(2+) channels, is profoundly inhibited. Furthermore, P2Y1 and P2Y6-mediated stimulation of migration is inhibited under conditions of extracellular acidosis, whereas basal migration independent of P2Y receptor activation is not. Taken together, our results demonstrate that an acidic microenvironment impacts on P2Y receptor-mediated Ca(2+) signalling, thereby influencing microglial responses and responsiveness to extracellular signals. This may result in altered behaviour of microglia under pathological conditions compared with microglial responses in healthy tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Reduction of hippocampal apoptosis by intracerebroventricular administration of extracellular signal-regulated protein kinase and/or p38 inhibitors in amyloid beta rat model of Alzheimer's disease: involvement of nuclear-related factor-2 and nuclear factor-κB.

    Science.gov (United States)

    Ashabi, Ghorbangol; Alamdary, Shabnam Zeighamy; Ramin, Mahmoudreza; Khodagholi, Fariba

    2013-03-01

    In the present study, we examined the effects of intracerebroventricular administration of extracellular signal-regulated protein kinase- (ERK) and p38-specific inhibitors, U0126 and PD169316, respectively, on apoptosis induced by amyloid beta (Aβ) in rats. To investigate the effects of these compounds, we evaluated intracellular signalling pathways of apoptosis, as well as inflammatory and antioxidant pathways, 7 and 20 days after Aβ injection. We found that caspase-3 and Bax/Bcl-2 ratio, two hallmarks of apoptosis, were significantly decreased in the rats pre-treated with U0126 and PD169316, 7 days after Aβ injection. This observation was in agreement with the results of immunostaining analysis of the hippocampus that showed decreased levels of terminal transferase dUTP nick end labelling positive cells in the hippocampus of U0126 and PD169316 pre-treated rats, compared with the Aβ-injected group. We also chased the changes in the levels of calpain-2 and caspase-12, two ER factors, in the Aβ-injected and treatment groups. Decreased levels of calpain-2 and caspase-12 in U0126 and PD169316 pre-treated rats confirmed the protective effects of these inhibitors. Furthermore, we studied the effect of two stress-sensing transcription factors, nuclear-related factor-2 (Nrf2) and nuclear factor-кB (NF-кB), in Aβ-injected as wells as U0126 and PD169316 pre-treated rats. U0126 and PD169316 activated Nrf2 and suppressed NF-кB pathways, 7 days after Aβ injection. These antioxidant and inflammatory pathways restored to the vehicle level within 20 days. Taken together, our findings reinforce and extend the notion of the potential neuroprotective role of ERK and/or p38 inhibitors against the neuronal toxicity induced by Aβ.

  19. Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells.

    Science.gov (United States)

    Mut, Melike; Lule, Sevda; Demir, Ozlem; Kurnaz, Isil Aksan; Vural, Imran

    2012-02-01

    Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.

  20. The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway.

    Science.gov (United States)

    Chandramohan, Yalini; Droste, Susanne K; Arthur, J Simon C; Reul, Johannes M H M

    2008-05-01

    The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

  1. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation.

    Science.gov (United States)

    Rossaint, Jan; Herter, Jan M; Van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

    2014-04-17

    There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute lung injury (ALI), which is associated with a high mortality rate in critically ill patients. NETs consist of decondensed chromatin decorated with granular and cytosolic proteins and they can trap extracellular pathogens. The prerequisite for NET formation is the activation of neutrophils and the release of their DNA. In a neutrophil- and platelet-dependent mouse model of ventilator-induced lung injury (VILI), NETs were found in the lung microvasculature, and circulating NET components increased in the plasma. In this model, blocking integrin-mediated outside-in or either GPCR-signaling or heteromerization of platelet chemokines decreased NET formation and lung injury. Targeting NET components by DNAse1 application or neutrophil elastase-deficient mice protected mice from ALI, whereas DNase1(-/-)/Trap1(m/m) mice had an aggravated ALI, suggesting that NETs directly influence the severity of ALI. These data suggest that NETs form in the lungs during VILI, contribute to the disease process, and thus may be a promising new direction for the treatment of ALI.

  2. Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4β7.

    Science.gov (United States)

    Plotnik, David; Guo, Wenjin; Cleveland, Brad; von Haller, Priska; Eng, Jimmy K; Guttman, Miklos; Lee, Kelly K; Arthos, James; Hu, Shiu-Lok

    2017-08-16

    Gut-homing α4β7(high) CD4(+) T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through α4β7, and that this likely contributes to the infection of α4β7(high) T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4β7 binding. However, lack of α4β7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4β7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4β7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4β7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4β7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion exchange chromatography abrogated V2-independent gp120-α4β7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4β7 interactions in a similar manner to CHO fibronectin. These findings provide an explanation for the apparent contradictory observations regarding the gp120-α4β7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV, and this plays an important role in the development of AIDS. Integrin α4β7 plays a major role in the trafficking of lymphocytes, including CD4(+) T cells, into gut lymphoid tissues. Previous reports

  3. Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Erin L. Foresman

    2013-01-01

    Full Text Available Superoxide (O2•− contributes to the development of cardiovascular disease. Generation of O2•− occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1 or extracellular SOD (SOD3 to blood vessels would differentially protect against O2•−-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad containing the gene for Escherichia coli β-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24 h following gene transfer. Histochemical staining for β-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05, whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control but not AdSOD1 (34±4%. We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2•− production in vascular disease.

  4. Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

    Directory of Open Access Journals (Sweden)

    Cristina Antonella Nadalutti

    Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

  5. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Xiao-Ping Chen; Wan-Guang Zhang; Feng Zhang; Shuai Xiang; Han-Hua Dong; Lei Zhang

    2009-01-01

    AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover.CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

  6. Sevoflurane postcondionting protects against focal cerebral ischemia/reperfusion injury via extracellular signaling-regulated kinase 1/2 pathway%七氟烷后处理通过激活细胞外信号调节激酶1/2信号通路减轻大鼠局灶性脑缺血/再灌注损伤

    Institute of Scientific and Technical Information of China (English)

    于颖波; 贾栋

    2014-01-01

    目的 研究七氟烷后处理对大鼠局部脑缺血/再灌注损伤(ischemia/reperfusion injury,I/RI)的影响,初步探讨细胞外信号调节激酶(extracellular signaling-regulated kinase,ERK)1/2信号通路在其中的作用. 方法 建立大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型.按随机数字表法将70只雄性Sprague-Dawley(SD)大鼠随机分为7组(每组10只):假手术组(Sham组)、I/RI组、七氟烷后处理组(Sevo组)、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/ERK1/2特异性阻断剂U0126组/PD98059组(U0126组/PD组)、七氟烷后处理+U0126组/PD98059组(Sevo+U0126组/Sevo+PD组).除Sham组外,其余各组大鼠均局部缺血90 min,再灌注24 h.Sevo组局部缺血90 min,再灌注开始即刻吸入2.5%七氟烷30 min行后处理,再灌注至24 h.特异性阻断剂U0126/PD98059分别于缺血前30 min显微镜下脑室内给予注射.再灌注24 h后行神经功能评分,测定脑梗死容积,Western blot法测定Total-ERK 1/2及磷酸化ERK1/2(ERK1/2 P42/P44)表达. 结果 与Sham组比较,I/RI组及Sevo组神经功能评分及脑梗死容积均增加(P<0.05);Sevo组神经功能评分及脑梗死容积较I/RI组显著降低[分别为(2.2±0.4)vs(3.2±0.6)和(24.1±2.1)%vs(42.3±2.2)%](P<0.05),磷酸化ERK1/2表达显著增高(P<0.05),U0126组、PD组及Sevo+U0126组、Sevo+PD组各指标组间比较差异无统计学意义(P>0.05). 结论 七氟烷后处理减轻大鼠局部脑I/RI,其作用机制可能与激活MAPK/ERK1/2信号通路有关.%Objective To investigate the effect of sevoflurane postconditioning on extracellular signaling-regulated kinase (ERK)1/2 after cerebral ischemia/reperfusion injury(I/RI) in rats.Methods Rat middle cerebral artery occlusion(MCAO) model was bulit.Seventy male Sprague-Dawley(SD) rats were randomly assigned to 7 groups (with 10 rats each):Sham group,I/RI group,sevoflurane postconditioning group (Sevo group),U0126/PD98059 [specific

  7. Effects of valproate sodium on extracellular signal-regulated kinase 1/2 phosphorylation after hippocampal neuronal epileptiform discharge in rats%丙戊酸钠对大鼠海马神经元癫痫样放电后细胞外信号调节激酶磷酸化水平的影响

    Institute of Scientific and Technical Information of China (English)

    徐祖才; 王学峰; 雷显泽; 徐忠祥; 徐平

    2012-01-01

    目的 细胞外信号调节激酶(extracellular signal-regulated kinase 1/2,ERK1/2)参与癫痫的发生,但其与抗癫痫药物之间的关系不明确,文中旨在观察丙戊酸钠对大鼠海马神经元癫痫样放电后磷酸化ERK1/2(p-ERK1/2)的影响.方法 取24h内新生Wistar大鼠,雌雄不拘,迅速断头取脑.建立神经元癫痫样放电模型,将神经元分为空白对照组和丙戊酸钠组,量效实验中,于神经元癫痫样放电前30min时加入不同浓度的丙戊酸钠(50mg/L、75mg/L、100mg/L),运用免疫荧光技术测定p-ERK1/2在不同浓度时的表达;时效实验中,分别于癫痫样放电前30min,放电后0min、30min、2h和6h加入50mg/L丙戊酸钠,采用 Wester blot观察p-ERK1/2的变化.结果 量效实验中,不同浓度的丙戊酸钠均能降低ERK1/2的磷酸化水平,且无显著性差异.时效实验中,于放电前30min时加入丙戊酸钠对ERK1/2的磷酸化水平抑制最明显,与以后各时间点间都有显著性差异.结论 海马神经元癫痫样放电后ERK1/2被过度持久的激活,在早期小剂量有效浓度的丙戊酸钠能显著抑制此反应中ERK1/2的磷酸化水平.%Objective Extracellular signal-regulated kinase l/2(ERKl/2) plays a role in the occurrence of epilepsy , but the mechanism of the involvement of ERK1/2 and its association with antiepileptic drugs remain unclear . The aim of this study is to investi -gate the effects of valproate sodium on ERK 1/2 phosphorylation (p-ERKl/2) after hippocampal neuronal epileptiform discharge in rats. Methods The epileptiform discharge model of the neuron was established in female and male neonate Wistar rats by rapid de -capitation. The neurons were divided into a blank control and a valproate sodium group , the latter incubated with valproate sodium at 50, 75 and 100 mg/L 30 min before epileptiform discharge in the concentration response experiment, and the expression of p-ERKl/2 at different concentrations detected using

  8. The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps.

    Directory of Open Access Journals (Sweden)

    Mark J Lee

    2015-10-01

    Full Text Available Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG, an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.

  9. Exogenous electron shuttle-mediated extracellular electron transfer of Shewanella putrefaciens 200: electrochemical parameters and thermodynamics.

    Science.gov (United States)

    Wu, Yundang; Liu, Tongxu; Li, Xiaomin; Li, Fangbai

    2014-08-19

    Despite the importance of exogenous electron shuttles (ESs) in extracellular electron transfer (EET), a lack of understanding of the key properties of ESs is a concern given their different influences on EET processes. Here, the ES-mediated EET capacity of Shewanella putrefaciens 200 (SP200) was evaluated by examining the electricity generated in a microbial fuel cell. The results indicated that all the ESs substantially accelerated the current generation compared to only SP200. The current and polarization parameters were linearly correlated with both the standard redox potential (E(ES)(0)) and the electron accepting capacity (EAC) of the ESs. A thermodynamic analysis of the electron transfer from the electron donor to the electrode suggested that the EET from c-type cytochromes (c-Cyts) to ESs is a crucial step causing the differences in EET capacities among various ESs. Based on the derived equations, both E(ES)(0) and EAC can quantitatively determine potential losses (ΔE) that reflect the potential loss of the ES-mediated EET. In situ spectral kinetic analysis of ES reduction by c-Cyts in a living SP200 suspension was first investigated with the E(ES), E(c-Cyt), and ΔE values being calculated. This study can provide a comprehensive understanding of the role of ESs in EET.

  10. Stromal fibroblasts mediate extracellular matrix remodeling and invasion of scirrhous gastric carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Hideki Yamaguchi

    Full Text Available Scirrhous gastric carcinoma (SGC has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.

  11. Extracellular haem peroxidases mediate Mn(II) oxidation in a marine Roseobacter bacterium via superoxide production.

    Science.gov (United States)

    Andeer, Peter F; Learman, Deric R; McIlvin, Matt; Dunn, James A; Hansel, Colleen M

    2015-10-01

    Manganese (Mn) oxides are among the strongest sorbents and oxidants in environmental systems. A number of biotic and abiotic pathways induce the oxidation of Mn(II) to Mn oxides. Here, we use a combination of proteomic analyses and activity assays, to identify the enzyme(s) responsible for extracellular superoxide-mediated Mn oxide formation by a bacterium within the ubiquitous Roseobacter clade. We show that animal haem peroxidases (AHPs) located on the outer membrane and within the secretome are responsible for Mn(II) oxidation. These novel peroxidases have previously been implicated in direct Mn(II) oxidation by phylogenetically diverse bacteria. Yet, we show that in this Roseobacter species, AHPs mediate Mn(II) oxidation not through a direct reaction but by producing superoxide and likely also by degrading hydrogen peroxide. These findings point to a eukaryotic-like oscillatory oxidative-peroxidative enzymatic cycle by these AHPs that leads to Mn oxide formation by this organism. AHP expression appears unaffected by Mn(II), yet the large energetic investment required to produce and secrete these enzymes points to an as yet unknown physiological function. These findings are further evidence that bacterial peroxidases and secreted enzymes, in general, are unappreciated controls on the cycling of metals and reactive oxygen species (ROS), and by extension carbon, in natural systems. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Ras Oncogene-Mediated Progressive Silencing of Extracellular Superoxide Dismutase in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Francesca Cammarota

    2015-01-01

    Full Text Available Extracellular superoxide dismutase (SOD3 is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reaction that results in the formation of hydrogen peroxide. Both of these reactive oxygen species affect growth signaling in cells. Although SOD3 has growth-supporting characteristics, the expression of SOD3 is downregulated in epithelial cancer cells. In the current work, we studied the mechanisms regulating SOD3 expression in vitro using thyroid cell models representing different stages of thyroid cancer. We demonstrate that a low level of RAS activation increases SOD3 mRNA synthesis that then gradually decreases with increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells. Our data indicate that SOD3 regulation can be divided into two classes. The first class involves RAS–driven reversible regulation of SOD3 expression that can be mediated by the following mechanisms: RAS GTPase regulatory genes that are responsible for SOD3 self-regulation; RAS-stimulated p38 MAPK activation; and RAS-activated increased expression of the mir21 microRNA, which inversely correlates with sod3 mRNA expression. The second class involves permanent silencing of SOD3 mediated by epigenetic DNA methylation in cells that represent more advanced cancers. Therefore, the work suggests that SOD3 belongs to the group of ras oncogene-silenced genes.

  13. RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

    Science.gov (United States)

    Jia, Daile; He, Yuhu; Zhu, Qian; Liu, Huan; Zuo, Caojian; Chen, Guilin; Yu, Ying; Lu, Ankang

    2017-05-01

    Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Se-Hee [McAllister Heart Institute, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Schmitt, Christopher E.; Woolls, Melissa J. [McAllister Heart Institute, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Yale Cardiovascular Research Center and Section of Cardiovascular Medicine, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511 (United States); Holland, Melinda B. [McAllister Heart Institute, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Kim, Jun-Dae [Yale Cardiovascular Research Center and Section of Cardiovascular Medicine, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511 (United States); Jin, Suk-Won, E-mail: suk-won.jin@yale.edu [Yale Cardiovascular Research Center and Section of Cardiovascular Medicine, Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06511 (United States)

    2013-01-25

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

  15. FGF signalling regulates bone growth through autophagy.

    Science.gov (United States)

    Cinque, Laura; Forrester, Alison; Bartolomeo, Rosa; Svelto, Maria; Venditti, Rossella; Montefusco, Sandro; Polishchuk, Elena; Nusco, Edoardo; Rossi, Antonio; Medina, Diego L; Polishchuk, Roman; De Matteis, Maria Antonietta; Settembre, Carmine

    2015-12-10

    Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.

  16. The Notch pathway mediates the angiotensin II-induced synthesis of extracellular matrix components in podocytes.

    Science.gov (United States)

    Yao, Min; Wang, Xiaomei; Wang, Xiaomeng; Zhang, Tao; Chi, Yanqing; Gao, Feng

    2015-07-01

    The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-β1 (TGF-β1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-β1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-β1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-β1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-β1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-β1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the

  17. Phosphatase of regenerating liver-3 promotes Lovo cells invasion by inducing serine protease inhibitor E3 through extracellular signal-regulated kinase%肝再生磷酸酶-3通过细胞外调节蛋白激酶上调丝氨酸蛋白酶抑制剂E3促进Lovo细胞侵袭

    Institute of Scientific and Technical Information of China (English)

    许鹤洋; 林显敢; 罗兴喜; 张旸; 蓝球生; 褚忠华

    2014-01-01

    Objective To investigate the influence and mechanisms of phosphatase of regenerating liver-3 (PRL-3) on serine protease inhibitor E3 (serpinE3),and their effects on Lovo cells invasion.Methods Western blotting was used to detect the serpinE3 of Lovo-P and Lovo-C cells.Transwell chamber was used to detect the invasion of Lovo-P and Lovo-C cells.The Lovo-P cells were treated with the extracellular signal-regulated kinase (ERK) inhibitor U0126 (10 μmol/L) for 6 h,and the expression of serpinE3 and invasion of Lovo-P cells were examined.Results The expression of serpinE3 was increased in the Lovo-P cells transfected with human PRL-3.Lovo-P cells exhibited stronger invasion ability than Lovo-Ccells (378 ± 13 vs.269 ± 15,P < 0.05).SerpinE3 was abrogated when Lovo-P treated with U0126 and the invasion ability of the cells was decreased either (211-±9 vs.358 ± 19,P <0.05).Conclusion PRL-3 could induce serpinE3 expression by ERK,and then promotes Lovo cells invasion.%目的 探讨肝再生磷酸酶-3(PRL-3)对丝氨酸蛋白酶抑制剂E3(serpinE3)的影响及机制,以及PRL-3和serpinF3对结肠癌Lovo细胞侵袭性的影响.方法 通过Western blot方法,分别检测已经稳转入PRL-3载体的Lovo-P细胞和对照组Lovo-C细胞中serpinE3的表达水平,Transwell小室检测Lovo细胞侵袭性.再给予细胞外调节蛋白激酶(ERK)特异性抑制剂U0126(10 μmol/L)预处理Lovo-P细胞6h,观察serpinE3表达的变化,检测Lovo-P细胞侵袭性的改变.结果 Western blot检测结果显示在转染人PRL-3的Lovo-P细胞中,serpinE3表达明显上调,Lovo-P细胞侵袭性增强[(378±13)个比(269±15)个,P<0.05];而当特异性阻断ERK后,Lovo-P细胞中的serpinE3表达下调,并且细胞侵袭性也降低[(21l±9)个比(358±19)个,P<0.05].结论 PRL-3能够通过ERK诱导serpinE3表达上调的方式,增加Lovo细胞的侵袭性.

  18. The relationship between effects of sorafenib on cell proliferation and basal phosphorylated extra-cellular signal-regulated kinase levels in different HCC cell lines%索拉非尼对人肝癌细胞的体外杀伤作用与磷酸化胞外信号调节激酶基础表达水平的关系

    Institute of Scientific and Technical Information of China (English)

    张哲; 王艳红; 周小芸; 王德兴; 沈沪佳

    2009-01-01

    Objective To investigate the relationship between effects of sorafenib on cell prolifer-ation and basal phosphorylated extracellular signal-regulated kinase (pERK) levels in different HCC cell lines. Methods pERK expression was quantitatively detected by immunocytochemistry and Western blot. The effects of various concentrations of sorafenib (0.01-30.00 μmol/L) on cell proliferation were evalua-ted by cell viability assay in four types of HCC cell line (SMMC-7721 ,MHCC97-L, MHCC97-H and HC-CLM6). Correlation analysis was carried out between the IC_(50) values of drugs and mean absorbance (A) values of pERK. Results The basal pERK levels were increased stepwise in cell lines in accordance with their metastatic potential. Correlation analysis between the IC_(50) values and mean A values of pERK re-vealed that the effects of sorafenib were significantly correlated with basal pERK levels (Spearman r =-0.8671 ,P <0. 01). Conclusion pERK was confirmed to be a useful biomarker predictive of sensitivity in treating HCC with sorafenib.%目的 探讨索拉非尼对不同肝细胞肝癌(HCC)胞株的体外杀伤作用与基础磷酸化胞外信号调节激酶(pERK)表达水平的关系.方法 应用细胞免疫化学定量分析和Western blot方法 检测方法 ,评价不同浓度(0.01~30.00 μmol/L)索拉非尼对4种人肝癌细胞(SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6)体外杀伤作用与细胞基础pERK表达的相关性.结果 4种细胞株基础pERK蛋白表达含量随着转移潜能依次递增.索拉非尼对各细胞株的IC_(50)与pERK蛋白表达呈负相关(Spearman r=-0.8671,P<0.01,n=12),提示索拉非尼的药物敏感性与细胞基础pERK蛋白水平存在显著的相关性.结论 pERK可以作为一个潜在的生物标记物,预测索拉非尼对肝细胞癌的药物敏感性.

  19. Extracellular vesicle-mediated delivery of molecular compounds into gametes and embryos: learning from nature.

    Science.gov (United States)

    Barkalina, Natalia; Jones, Celine; Wood, Matthew J A; Coward, Kevin

    2015-01-01

    Currently, even the most sophisticated methods of assisted reproductive technology (ART) allow us to achieve live births in only approximately 30% of patients, indicating that our understanding of the fine mechanisms underlying reproduction is far from ideal. One of the main challenges associated with studies of gamete structure and function is that these cells are remarkably resistant towards the uptake of exogenous substances, including 'molecular research tools' such as drugs, biomolecules and intracellular markers. This phenomenon can affect not only the performance of reproductive biology research techniques, but also the outcomes of the in vitro handling of gametes, which forms the cornerstone of ART. Improvement of intra-gamete delivery in a non-aggressive fashion is vital for the investigation of gamete physiology, and the advancement of infertility treatment. In this review, we outline the current state of nanomaterial-mediated delivery into gametes and embryos in vitro, and discuss the potential of a novel exciting drug delivery technology, based upon the use of targeted 'natural' nanoparticles known as extracellular vesicles (EVs), for reproductive science and ART, given the promising emerging data from other fields. A comprehensive electronic search of PubMed and Web of Science databases was performed using the following keywords: 'nanoparticles', 'nanomaterials', 'cell-penetrating peptides', 'sperm', 'oocyte', 'egg', 'embryo', 'exosomes', 'microvesicles', 'extracellular vesicles', 'delivery', 'reproduction', to identify the relevant research and review articles, published in English up to January 2015. The reference lists of identified publication were then scanned to extract additional relevant publications. Biocompatible engineered nanomaterials with high loading capacity, stability and selective affinity represent a potential versatile tool for the minimally invasive internalization of molecular cargo into gametes and embryos. However, it is

  20. Extracellular signal-regulated kinase signaling pathway regulates the endothelial differentiation of periodontal ligament stem cells%细胞外调节蛋白激酶信号通路调控牙周膜干细胞内皮分化的机制研究

    Institute of Scientific and Technical Information of China (English)

    朱宏; 罗兰堃; 王颖; 谈珺; 薛芃; 王勤涛

    2016-01-01

    目的 探讨细胞外调节蛋白激酶(extracellular signal-regulated kinase,ERK)通路在牙周膜干细胞(periodontal ligament stem cells,PDLSC)内皮向分化中的作用,为PDLSC分化调控提供实验基础.方法 使用血管内皮生长因子(vascular endothelial growth factor,VEGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,b-FGF)联合诱导PDLSC向内皮细胞分化(诱导组),对诱导分化的细胞使用ERK1/2磷酸化阻断剂U0126进行处理(诱导+U0126组),同时用二甲基亚砜(dimethylsulfoxide,DMSO)作为对照(诱导+DMSO组),空白对照组为未经诱导的PDLSC;蛋白质印迹法检测诱导组0、1、3、6及12h的胞内磷酸化ERK1/2 (p-ERK1/2)表达水平;各组诱导7d后提取细胞RNA,实时荧光定量PCR法检测细胞内CD31、血管内皮钙黏素(vascular endothelial-cadherin,VE-cadherin)和VEGF mRNA的表达情况;各组诱导14d,流式细胞计数法检测CD31+和VE-cadherin+细胞比例,基质胶管腔形成实验检测细胞的管腔形成能力.计量资料采用均值±标准差表示,两组间比较采用独立样本t检验;多组间比较采用单因素方差分析.结果 诱导1、3h后p-ERK1/2与ERK1/2比值分别升高至1.24±0.12、1.03±0.24,均显著高于诱导前(0.58±0.17)(P<0.01);实时荧光定量PCR结果显示,诱导+U0126组CD31、VEGF mRNA相对表达水平分别降至0.09±0.18、0.49±0.17,均显著低于诱导组细胞(P<0.05);流式细胞检测结果显示,诱导+U0126组CD31+、VE-cadherin+细胞比值分别降至5.22±0.85、3.56±0.87,均显著低于诱导组细胞(P<0.05);基质胶管腔形成实验显示诱导组的分支节点数、管腔数目、管腔长度分别升高至62.3±10.0、145.0±14.8及(32 129.7±4 413.9)像素,而诱导+U0126组分别下降至7.0±2.7、33.5±6.4及(15 951.0±758.1)像素,均显著低于诱导组(P<0.05).结论 PDLSC内皮分化过程受ERK通路正向调控,阻断ERK1/2磷酸化可以

  1. Effect of pERK2 on extracellular matrix turnover of the fibrotic joint capsule in a post-traumatic joint contracture model

    OpenAIRE

    Sun, Yangbai; Li, Fengfeng; Fan, Cunyi

    2015-01-01

    Lentivirus (LV)-mediated extracellular signal-regulated kinase (ERK)2 small interfering RNA (siRNA) has previously been demonstrated to reduce post-traumatic joint contractures: In the present study, the effect of ERK2 siRNA on extracellular matrix turnover within fibrotic joint capsules in post-traumatic joint contractures was examined. Rats were randomly assigned to one of three groups as follows: The non-operated control (CON), operated contracture (ORC) and contracture-treatment (CNT) gro...

  2. Extracellular polymeric substances mediate bioleaching/biocorrosion via interfacial processes involving iron(III) ions and acidophilic bacteria.

    Science.gov (United States)

    Sand, Wolfgang; Gehrke, Tilman

    2006-01-01

    Extracellular polymeric substances seem to play a pivotal role in biocorrosion of metals and bioleaching, biocorrosion of metal sulfides for the winning of precious metals as well as acid rock drainage. For better control of both processes, the structure and function of extracellular polymeric substances of corrosion-causing or leaching bacteria are of crucial importance. Our research focused on the extremophilic bacteria Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, because of the "simplicity" and knowledge about the interactions of these bacteria with their substrate/substratum and their environment. For this purpose, the composition of the corresponding extracellular polymeric substances and their functions were analyzed. The extracellular polymeric substances of both species consist mainly of neutral sugars and lipids. The functions of the exopolymers seem to be: (i) to mediate attachment to a (metal) sulfide surface, and (ii) to concentrate iron(III) ions by complexation through uronic acids or other residues at the mineral surface, thus, allowing an oxidative attack on the sulfide. Consequently, dissolution of the metal sulfide is enhanced, which may result in an acceleration of 20- to 100-fold of the bioleaching process over chemical leaching. Experiments were performed to elucidate the importance of the iron(III) ions complexed by extracellular polymeric substances for strain-specific differences in oxidative activity for pyrite. Strains of A. ferrooxidans with a high amount of iron(III) ions in their extracellular polymeric substances possess greater oxidation activity than those with fewer iron(III) ions. These data provide insight into the function of and consequently the advantages that extracellular polymeric substances provide to bacteria. The role of extracellular polymeric substances for attachment under the conditions of a space station and resulting effects like biofouling, biocorrosion, malodorous gases, etc. will be discussed.

  3. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Theerthankar Das

    Full Text Available Calcium (Ca(2+ has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG values in iTC data confirmed that the interaction between DNA and Ca(2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+ to eDNA thereby mediating bacterial aggregation and biofilm formation.

  4. Aflatoxin B1 Induces Reactive Oxygen Species-Mediated Autophagy and Extracellular Trap Formation in Macrophages

    Science.gov (United States)

    An, Yanan; Shi, Xiaochen; Tang, Xudong; Wang, Yang; Shen, Fengge; Zhang, Qiaoli; Wang, Chao; Jiang, Mingguo; Liu, Mingyuan; Yu, Lu

    2017-01-01

    Aflatoxins are a group of highly toxic mycotoxins with high carcinogenicity that are commonly found in foods. Aflatoxin B1 (AFB1) is the most toxic member of the aflatoxin family. A recent study reported that AFB1 can induce autophagy, but whether AFB1 can induce extracellular traps (ETs) and the relationships among innate immune responses, reactive oxygen species (ROS), and autophagy and the ETs induced by AFB1 remain unknown. Here, we demonstrated that AFB1 induced a complete autophagic process in macrophages (MΦ) (THP-1 cells and RAW264.7 cells). In addition, AFB1 induced the generation of MΦ ETs (METs) in a dose-dependent manner. In particular, the formation of METs significantly reduced the AFB1 content. Further analysis using specific inhibitors showed that the inhibition of either autophagy or ROS prevented MET formation caused by AFB1, indicating that autophagy and ROS were required for AFB1-induced MET formation. The inhibition of ROS prevented autophagy, indicating that ROS generation occurred upstream of AFB1-induced autophagy. Taken together, these data suggest that AFB1 induces ROS-mediated autophagy and ETs formation and an M1 phenotype in MΦ. PMID:28280716

  5. β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps.

    Science.gov (United States)

    Raftery, Martin J; Lalwani, Pritesh; Krautkrӓmer, Ellen; Peters, Thorsten; Scharffetter-Kochanek, Karin; Krüger, Renate; Hofmann, Jörg; Seeger, Karl; Krüger, Detlev H; Schönrich, Günther

    2014-06-30

    Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin-mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage.

  6. Differential expression of extracellular matrix-mediated pathways in single-suture craniosynostosis.

    Directory of Open Access Journals (Sweden)

    Brendan D Stamper

    Full Text Available Craniosynostosis is a disease defined by premature fusion of one or more cranial sutures. The mechanistic pathology of single-suture craniosynostosis is complex and while a number of genetic biomarkers and environmental predispositions have been identified, in many cases the causes remain controversial and inconclusive. In this study, gene expression data from 199 patients with isolated sagittal (n = 100, unilateral coronal (n = 50, and metopic (n = 49 synostosis are compared against both a control population (n = 50, as well as each other. After controlling for variables contributing to potential bias, FGF7, SFRP4, and VCAM1 emerged as genes associated with single-suture craniosynostosis due to their significantly large changes in gene expression compared to the control population. Pathway analysis implicated focal adhesion and extracellular matrix (ECM-receptor interaction as differentially regulated gene networks when comparing all cases of single-suture synostosis and controls. Lastly, overall gene expression was found to be highly conserved between coronal and metopic cases, as evidenced by the fact that WNT2 and IGFBP2 were the only genes differentially regulated to a significantly large extent in a direct comparison. The identification of genes and gene networks associated with Fgf/Igf/Wnt signaling and ECM-mediated focal adhesion not only support the involvement of biomarkers previously reported to be related to craniosynostosis, but also introduce novel transcripts and pathways that may play critical roles in its pathogenesis.

  7. Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen

    Science.gov (United States)

    Szatmári, Tünde; Kis, Dávid; Bogdándi, Enikő Noémi; Benedek, Anett; Bright, Scott; Bowler, Deborah; Persa, Eszter; Kis, Enikő; Balogh, Andrea; Naszályi, Lívia N.; Kadhim, Munira; Sáfrány, Géza; Lumniczky, Katalin

    2017-01-01

    Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation

  8. The minimal essential unit for cadherin-mediated intercellular adhesion comprises extracellular domains 1 and 2

    DEFF Research Database (Denmark)

    Shan, Weisong; Yagita, Yoshiki; Wang, Zhaohui

    2004-01-01

    N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the ...

  9. An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ermakov, Aleksei V., E-mail: avePlato@mail.ru [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Konkova, Marina S.; Kostyuk, Svetlana V.; Smirnova, Tatiana D.; Malinovskaya, Elena M.; Efremova, Liudmila V.; Veiko, Natalya N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2011-07-01

    The human umbilical vein endothelial cells culture was exposed to X-ray radiation in a low dose of 10 cGy. The fragments of extracellular genomic DNA (ecDNA{sup R}) were isolated from the culture medium after the short-term incubation. A culture medium of unirradiated endothelial cells was then supplemented with ecDNA{sup R}, followed by analysing the cells along the series of parameters (bystander effect). The exposed cells and bystander endotheliocytes showed similar response to low doses: approximation of the 1q12 loci of chromosome 1 and their transposition into the cellular nucleus, change in shape of the endotheliocytic nucleus, activation of the nucleolus organizing regions (NORs), actin polymerization, and an elevated level of DNA double-stranded breaks. Following blockade of TLR9 receptors with oligonucleotide-inhibitor or chloroquine in the bystander cells these effects - except of activation of NORs - on exposure to ecDNA{sup R} disappeared, with no bystander response thus observed. The presence of the radiation-induced apoptosis in the bystander effect being studied suggests a possibility for radiation-modified ecDNA fragments (i.e., stress signaling factors) to be released into the culture medium, whereas inhibition of TLR9 suggests the binding these ligands to the recipient cells. A similar DNA-signaling pathway in the bystander effect we previously described for human lymphocytes. Integrity of data makes it possible to suppose that a similar signaling mechanism which we demonstrated for lymphocytes (humoral system) might also be mediated in a monolayer culture of cells (cellular tissue) after the development of the bystander effect in them and transfer of stress signaling factors (ecDNA{sup R}) through the culture medium.

  10. An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells.

    Science.gov (United States)

    Ermakov, Aleksei V; Konkova, Marina S; Kostyuk, Svetlana V; Smirnova, Tatiana D; Malinovskaya, Elena M; Efremova, Liudmila V; Veiko, Natalya N

    2011-07-01

    The human umbilical vein endothelial cells culture was exposed to X-ray radiation in a low dose of 10cGy. The fragments of extracellular genomic DNA (ecDNA(R)) were isolated from the culture medium after the short-term incubation. A culture medium of unirradiated endothelial cells was then supplemented with ecDNA(R), followed by analysing the cells along the series of parameters (bystander effect). The exposed cells and bystander endotheliocytes showed similar response to low doses: approximation of the 1q12 loci of chromosome 1 and their transposition into the cellular nucleus, change in shape of the endotheliocytic nucleus, activation of the nucleolus organizing regions (NORs), actin polymerization, and an elevated level of DNA double-stranded breaks. Following blockade of TLR9 receptors with oligonucleotide-inhibitor or chloroquine in the bystander cells these effects - except of activation of NORs - on exposure to ecDNA(R) disappeared, with no bystander response thus observed. The presence of the radiation-induced apoptosis in the bystander effect being studied suggests a possibility for radiation-modified ecDNA fragments (i.e., stress signaling factors) to be released into the culture medium, whereas inhibition of TLR9 suggests the binding these ligands to the recipient cells. A similar DNA-signaling pathway in the bystander effect we previously described for human lymphocytes. Integrity of data makes it possible to suppose that a similar signaling mechanism which we demonstrated for lymphocytes (humoral system) might also be mediated in a monolayer culture of cells (cellular tissue) after the development of the bystander effect in them and transfer of stress signaling factors (ecDNA(R)) through the culture medium. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Exchange of extracellular domains of CCR1 and CCR5 reveals confined functions in CCL5-mediated cell recruitment.

    Science.gov (United States)

    Kramp, Birgit K; Megens, Remco T A; Sarabi, Alisina; Winkler, Sabine; Projahn, Delia; Weber, Christian; Koenen, Rory R; von Hundelshausen, Philipp

    2013-10-01

    The chemokine CCL5 recruits monocytes into inflamed tissues by triggering primarily CCR1-mediated arrest on endothelial cells, whereas subsequent spreading is dominated by CCR5. The CCL5-induced arrest can be enhanced by heteromer formation with CXCL4. To identify mechanisms for receptor-specific functions, we employed CCL5 mutants and transfectants expressing receptor chimeras carrying transposed extracellular regions. Mutation of the basic 50s cluster of CCL5, a coordinative site for CCL5 surface presentation, reduced CCR5- but not CCR1-mediated arrest and transmigration. Impaired arrest was restored by exchanging the CCR5-N-terminus for that of CCR1, which supported arrest even without the 50s cluster, whereas mutation of the basic 40s cluster essential for proteoglycan binding of CCL5 could not be rescued. The enhancement of CCL5-induced arrest by CXCL4 was mediated by CCR1 requiring its third extracellular loop. The domain exchanges did not affect formation and co-localisation of receptor dimers, indicating a sensing role of the third extracellular loop for hetero-oligomers in an arrest microenvironment. Our data identify confined targetable regions of CCR1 specialised to facilitate CCL5-induced arrest and enhanced responsiveness to the CXCL4-CCL5 heteromer.

  12. Role of extracellular signal-regulated kinase in central amygdala on fentanyl-induced hyperalgesia in rats%中央杏仁核细胞外信号调节激酶在芬太尼诱发大鼠痛觉过敏中的作用

    Institute of Scientific and Technical Information of China (English)

    尹平平; 葛胜辉; 罗放

    2015-01-01

    目的 探讨中央杏仁核细胞外信号调节激酶(ERK)在芬太尼诱发大鼠痛觉过敏中的作用.方法 清洁级健康雄性SD大鼠32只,体重60~ 100 g,采用随机数字表法分为4组(n=8):对照组(C组)皮下注射生理盐水,6.5 h后中央杏仁核导管注射DMSO;芬太尼诱发痛觉过敏组(H组)皮下注射芬太尼制备模型,6.5 h后中央杏仁核导管注射DMSO;ERKl抑制剂U0124组(U1组)制备模型,6.5 h后中央杏仁核导管注射U0124 1.5 nmol;ERK1/2抑制剂U0126组(U2组)制备模型,6.5h后中央杏仁核导管注射U0126 1.5 nmol.于注射芬太尼前、注射后6.5h和导管内给药后30 min(T0-2)时测定机械痛阈和热痛阈,随后C组和H组处死大鼠,取中央杏仁核组织采用Western blot法检测磷酸化ERK1/2(p-ERK1/2)的表达.结果 与C组比较,H组和U1组T1.2时、U2组T1时机械痛阈和热痛阈降低,H组中央杏仁核p-ERK2表达上调(P<0.05),p-ERK1表达差异无统计学意义(P>0.05);与H组比较,U2组T2时机械痛阈和热痛阈升高(P<0.05),U1组机械痛阈和热痛阈差异无统计学意义(P>0.05).结论 中央杏仁核ERK2激活参与了芬太尼诱发大鼠痛觉过敏的形成过程.%Objective To investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) in the central amygdala on fentanyl-induced hyperalgesia in rats.Methods Thirty-two male SpragueDawley rats, weighing 60-100 g, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C), fentanyl-induced hyperalgesia group (group H), U0124 group (group U1) , and U0126 group (group U2).A catheter was implanted in the central amygdale.In group C, normal saline was injected subcutaneously, and 6.5 h later dimethyl sulfoxide (DMSO) was injected via the catheter.In group H, fentanyl was injected subcutaneously to induce hyperalgesia, and 6.5 h later DMSO was injected via the catheter.In group U1, hyperalgesia was induced, and 6.5 h later ERK1 inhibitor U0124 1

  13. 细粒棘球蚴细胞外信号调节激酶基因克隆、序列分析及功能的初步鉴定%Molecular cloning, sequencing and function of extracellular signal regulated kinase of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    吕国栋; 纪静; 王俊华; 李亮; 王红丽; 卢晓梅; 王星; 温浩; 林仁勇

    2010-01-01

    Objective To perform molecular cloning and sequencing, bioinformatics analysis,protein expression and function of extracellular signal regulated kinase (EgERK1) of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and prokaryotic expression plasmid pET28a-EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34.This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase.Western blot results showed the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.%目的 从新疆株细粒棘球蚴中克隆细胞外信号调节激酶(EgERK1)基因,进行序列测定、生物信息学分析,蛋白表达及功能初步鉴定.方法 设计特异性引物,从新疆株细粒棘球蚴中提取总RNA,RT-PCR法扩增EgERK1基因,构建pET28

  14. Role of extracellular signal-regulated kinases in aldosterone-induced rat mesangial cells proliferation%细胞外信号调节蛋白激酶介导醛固酮诱导的肾小球系膜细胞增殖

    Institute of Scientific and Technical Information of China (English)

    姚丽; 孙立; 魏敏; 葛丹梅; 王力宁

    2011-01-01

    目的 探讨细胞外信号调节蛋白激酶(ERK1/2)在醛固酮诱导肾小球系膜细胞(RMC)增殖中的作用.方法 获取6~8周健康雄性SD大鼠RMC并鉴定,取第5~10代的细胞用于实验.细胞分为6组:对照组;PD98059(10 μmol/L)组;依普利酮(1 μmol/L)组;醛固酮(100 nmol/L)组;醛固酮(100 nmol/L)+PD98059(10 μmol/L)组;醛固酮(100 nmol/L)+依普利酮(1 μmol/L)组.采用Western印迹技术检测SD大鼠RMC盐皮质激素受体(MR)表达状况,以及醛固酮刺激后RMC ERK1/2活性状态.采用3H-胸腺嘧啶核苷(3H-TdR)掺入法检测RMC增殖状况.结果 体外培养的SD大鼠RMC有MR蛋白表达.醛固酮(100 nmol/L)刺激RMC 10 min使ERK1/2活性显著增高(P<0.05);刺激30 h时使RMC的3H-TdR掺入量显著增加[(1.35±0.08)倍,P<0.05].选择性醛固酮受体拮抗剂依普利酮(1 μmol/L)及MAPKERK激酶(MEK)特异性抑制剂PD98059(10 μmol/L)可阻止醛固酮诱导的RMC ERK1/2激活以及3H-TdR掺入量增加.结论 醛固酮通过激活ERK1/2信号转导通路诱导RMC增殖.%Objective To determine the role of extracellular signal-regulated kinases (ERK1/2) in aldosterone-induced rat mesangial cells (RMCs) proliferation. Methods RMCs were obtained from intact glomeruli of 4- to 6-week-old Sprague-Dawley rats and characterized according to published methods. RMCs between passages 5 and passages 10 were used. Protein levels of mineralocorticoid receptor (MR) in RMCs were analyzed by Western blotting. The cells were divided into the following groups: control group, PD98059 (10 (μmol/L) group, eplerenone (1 μmol/L) group, aldosterone (100 nmol/L) group, aldosterone (100 nmol/L) +PD98059 (10 μmol/L) group, aldosterone (100 nmol/L)+eplerenone (1 μmol/L) group. ERK1/2 activity was measured by Western blotting. Cell proliferation of RMCs was evaluated by [3H]-thymidine uptake measurements.Results MR protein expression in RMCs was confirmed by Western blotting. Aldosterone activated ERK1/2, and the maximal

  15. Role of extracellular signal-regulated kinase in neuronal apoptosis of hippocampns induced by global cerebral ischemia-reperfusion in rats%ERK在脑缺血再灌注大鼠海马细胞凋亡中的作用

    Institute of Scientific and Technical Information of China (English)

    何家璇; 薛荣亮; 吴刚

    2008-01-01

    目的 探讨细胞外信号调节激酶(ERK)在脑缺血再灌注大鼠海马细胞凋亡中的作用.方法 健康雄性SD大鼠90只,体重280~320 g,随机分为3组(n=30):假手术组(S组)、脑缺血再灌注组(IR组)和ERK磷酸化特异性抑制剂PD98059组(PD组).采用4血管法建立大鼠脑缺血再灌注模型,于再灌注后2、6、12、24、48、72 h时,各取5只大鼠,断头取脑,光镜下观察海马CA1区和CA3区病理学结果 ,计算细胞凋亡指数(AI),采用免疫组化法检测磷酸化ERK(p-ERK)和磷酸化Bad(p-Bad)的表达.结果 与S组比较,IR组和PD组再灌注期间CAI区和CA3区AI升高,再灌注2、6、12 h时CA1区p-ERK表达降低,再灌注后CA1区和CA3区p-Bad表达降低(P<0.05);与IR组比较,PD组再灌注后CA1医和CA3区AI升高,再灌注2、6、12、24 h时CA3区p-ERK表达降低,再灌注2、6 h时CA1区p-Bad表达降低,再灌注2、6、12h时CA3区p-Bad表达降低(P<0.05).结论 脑缺血再灌注可降低ERK活性,导致Bad蛋白去磷酸化,从而诱发大鼠海马细胞凋亡.%Objective To investigate the role of extracellular signal-regulated kinase in neuronal apoptosis of hippoeampus induced by global cerebral ischemia-reperfusion in rats.Methods Ninety healthy male SD rats weighing 280-320 g were randomly divided into 3 groups(n=30 each):groupI sham operation(S);groupⅡI/ R and group Ⅲ PD98059+I/R(PD).The animals were anesthetized with intraperitoneal 1% pentobarbital 40 mgkg.Global cerebral I/R was produced by 4-vessel occlusion method.Bilateral vertebral arteries were electrically cauterized and bilateral common carotid arteries were clamped for 5 min.Clamping was then released for reperfusion in group Ⅱ and Ⅲ.In group Ⅲ PD98059(a specific ERK inhibitor)O.3 mg/kg was injected iv before carotid artery clamping.Five animals in each group were sacrificed at 2,6,12,24,48 and 72 h of reperfusion and their brains were removed and cut into sections which were stained with HE and examined

  16. Extracellular signal-regulated protein kinase activation in endometrium with polycystic ovary syndrome and its significance%细胞外信号调节激酶在多囊卵巢综合征患者子宫内膜中的活化及意义

    Institute of Scientific and Technical Information of China (English)

    宋学茹; 张慧英; 张艳芳; 韩玉崑; 李可君

    2010-01-01

    Objective To investigate the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling pathway in the endometrium of women with polycystic ovary syndrome (PCOS) and its effect and significance in the cause of hyperplasia and carcinoma;and investigate the factors which affect the activation of the MAPK/ERK signaling pathway. Methods Collected 52 patients diagnosed as PCOS who were taken dilation and curettage of uterus as study, while 32 non-PCOS patients matched as control group. Serum hormonal parameters, fasting blood glucose and insulin were measured in all patients. The PCOS patients were sub-group as insulin resistance group and non-insulin resistance group; all the patients were carried out pathology inspection of endometria, and the PCOS patients were sub-group as endometrial hyperplasia and carcinoma group and normal endometrium group based on the outcome of pathology inspection. Western blot were performed to detect the expressions of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2), the activation of ERK1/2. Results (1)The expression of pERK1/2 [(61 ±13)%] in the endometrium in PCOS group was higher than that in the control [(44 ±10)%, P <0.01]. (2)The expression of p-ERK1/2 was significantly increased in group of endometrial hyperplasia and carcinoma [ ( 70 ± 11 )% ] compared to that in group of normal endometrium [ (55 ± 10)% ,P < 0.01 ], while there were significant difference between group of insulin resistance [ (63 ± 13 )% ] and group of non-insulin resistance [ (55 ±7)%, P <0.01 ]. (3) Fasting insulin level, insulin area under the curve and body mass index were related to the expression of p-ERK1/2 in endometrium with PCOS, the correlation coefficient were 0.447, 0.456 and 0.381, respectively ( all P < 0.01 ). Conclusions The MAPK/ERK signaling pathway in endometrium with PCOS was overactivation, which was related to the endometrial hyperplasia and carcinoma; while the activation of

  17. Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

    Science.gov (United States)

    Heck, Jessica N.; Ponik, Suzanne M.; Garcia-Mendoza, Maria G.; Pehlke, Carolyn A.; Inman, David R.; Eliceiri, Kevin W.; Keely, Patricia J.

    2012-01-01

    Breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. In turn, matrix stiffness regulates RhoA activity. However, the upstream signaling mechanisms are poorly defined. Here we demonstrate that the Rho exchange factor GEF-H1 mediates RhoA activation in response to extracellular matrix stiffness. We demonstrate the novel finding that microtubule stability is diminished by a stiff three-dimensional (3D) extracellular matrix, which leads to the activation of GEF-H1. Surprisingly, activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway did not contribute to stiffness-induced GEF-H1 activation. Loss of GEF-H1 decreases cell contraction of and invasion through 3D matrices. These data support a model in which matrix stiffness regulates RhoA through microtubule destabilization and the subsequent release and activation of GEF-H1. PMID:22593214

  18. Involvement of extracellular signal-regulated kinases 1/2 phosphorylation in estrogen-modulated nociception in rats with incision pain%磷酸化胞外信号调节激酶1/2参与雌激素对切口痛大鼠的伤害性感受调节

    Institute of Scientific and Technical Information of China (English)

    姚婧鑫; 赵欣; 薛庆生; 于布为

    2011-01-01

    Objective To investigate the role of extracellular signal-regulated kinases pERK1/2 phosphorylation in estrogen-modulated nociception of incision pain in rats. Methods Thirty two adult ovariectomized (OVX) female rats were used in this study. The plantar incision operation was performed on the 15th day after OVX. OVX rats were divided into 4 groups, with 8 in each group. Group E + S: estrogen replacement (50 μg estrogen dissolved in 100 uL olive oil) + sham incision; group V+ S: vehicle replacement (100 μL olive oil) + sham incision; group E+l: estrogen replacement (50 pg estrogen dissolved in 100 μL olive oil) + incision operation; and group V+l: vehicle replacement (100 μL olive oil) + incision operation. Estrogen or vehicle replacements were intraperitoneally injected every other day from day 14 after OVX to the end of pain behavior test. Paw withdrawal thermal latency (PWTL) assessment was used as the pain behavior test before OVX, 2 day before paw incision operation, and, 1,3,5,7 days after incision. ERK1/2 phosphorylation (pERKI/ 2) was assayed in spinal dorsal cord after pain behavior test. Results Compared with group E + S, PWTL in group V+S were significantly increased on day 16, 18, 20, 22 after OVX (P<0.05). The PWTL at 1 and 3 daysafter incision operation were significant lower than the PWTL before incision in group E+ I and group V+ I. The PWTL of rats in group E+ I were significantly reduced compared with that in group V+ I on the day 1 and 3 after incision operation. The PWTL of group E+ I and group V+ I reverted to the preoperative levels of incision pains on the day 5 and day 7 postoperatively, respectively. pERK1/2 expression in the spinal cord of rats in estrogen replacement groups were augmented more than those in the vehicle groups. After incision operation* the level of pERK1/2 in the ipsilateral side was higher than that in the contralateral side of spinal cord (P<0. 05). Conclusion Estrogen can increase the nociception of OVX incision

  19. Intracellular Transactivation of Epidermal Growth Factor Receptor by alpha(1A)-Adrenoceptor Is Mediated by Phosphatidylinositol 3-Kinase Independently of Activation of Extracellular Signal Regulated Kinases 1/2 and Serine-Threonine Kinases in Chinese Hamster Ovary Cells

    NARCIS (Netherlands)

    Ulu, Nadir; Henning, Robert H.; Guner, Sahika; Zoto, Teuta; Duman-Dalkilic, Basak; Duin, Marry; Gurdal, Hakan

    2013-01-01

    Transactivation of epidermal growth factor receptor (EGFR) by alpha(1)-adrenoceptor (alpha(1)-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all alpha(1)-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster

  20. Flavins mediate extracellular electron transfer in Gram-positive Bacillus megaterium strain LLD-1

    DEFF Research Database (Denmark)

    Xiao, Yong; Liu, Lidan; You, Lexing;

    electrochemically active strain of Bacillus megatherium strain LLD-1, and its extracellular electron transfer mechanism was demonstrated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), HPLC, and chronoamperometric. The CV and DPV showed that a redox peaks ascribing to membrane proteins was found...

  1. The concentration of extracellular superoxide dismutase in plasma is maintained by LRP-mediated endocytosis

    DEFF Research Database (Denmark)

    Petersen, Steen V; Thøgersen, Ida B; Valnickova, Zuzana;

    2010-01-01

    In this study, we show that human extracellular superoxide dismutase (EC-SOD) binds to low-density lipoprotein receptor-related protein (LRP). This interaction is most likely responsible for the removal of EC-SOD from the blood circulation via LRP expressed in liver tissue. The receptor recogniti...

  2. In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior

    NARCIS (Netherlands)

    Zomer, Anoek; Maynard, Carrie; Verweij, Frederik Johannes; Kamermans, Alwin; Schafer, Ronny; Beerling, Evelyne; Schiffelers, Raymond Michel; de Wit, Elzo; Berenguer, Jordi; Ellenbroek, Saskia Inge Johanna; Wurdinger, Thomas; Pegtel, Dirk Michiel; van Rheenen, Jacco

    2015-01-01

    Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challengin

  3. Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

    DEFF Research Database (Denmark)

    Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

    2003-01-01

    for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions......Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix...

  4. Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

    Directory of Open Access Journals (Sweden)

    Chen Zhang

    2016-09-01

    Full Text Available Ca2+-sensing receptors (CaSRs play a central role in regulating extracellular calcium concentration ([Ca2+]o homeostasis and many (pathophysiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

  5. Extracellular polysaccharides mediate pilus retraction during social motility of Myxococcus xanthus.

    Science.gov (United States)

    Li, Yinuo; Sun, Hong; Ma, Xiaoyuan; Lu, Ann; Lux, Renate; Zusman, David; Shi, Wenyuan

    2003-04-29

    Myxococcus xanthus is a Gram-negative bacterium with a complex life cycle that includes vegetative swarming and fruiting-body formation. Social (S)-motility (coordinated movement of large cell groups) requires both type IV pili and fibrils (extracellular matrix material consisting of polysaccharides and protein). Little is known about the role of this extracellular matrix, or fibril material, in pilus-dependent motility. In this study, mutants lacking fibril material and, therefore, S-motility were found to be hyperpiliated. We demonstrated that addition of fibril material resulted in pilus retraction and rescued this phenotype. The fibril material was further examined to determine the component(s) that were responsible for triggering pilus retraction. Protein-free fibril material was found to be highly active in correcting hyperpiliation. However, the amine sugars present in hydrolyzed fibril material, e.g., glucosamine and N-acetylglucosamine (GlcNAc) had no effect on fibril(-) mutants, but, interestingly, cause hyperpiliation in wild-type cells. In contrast, chitin, a natural GlcNAc polymer, was found to restore pilus retraction in hyperpiliated mutants, indicating that a polysaccharide containing amine sugars is likely required for pilus retraction. These data suggest that the interaction of type IV pili with amine-containing polysaccharides on cell and slime-trail surfaces may trigger pilus retraction, resulting in S-motility and slime-trailing behaviors.

  6. Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

    Science.gov (United States)

    Bridgen, D T; Gilchrist, C L; Richardson, W J; Isaacs, R E; Brown, C R; Yang, K L; Chen, J; Setton, L A

    2013-10-01

    The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

  7. Analysis of neofructooligosaccharides production mediated by the extracellular β-fructofuranosidase from Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Linde, Dolores; Rodríguez-Colinas, Barbara; Estévez, Marta; Poveda, Ana; Plou, Francisco J; Fernández Lobato, María

    2012-04-01

    The extracellular β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous mainly synthesizes the neo-fructooligosaccharides (neo-FOS) neokestose and neonystose. This enzyme is a glycoprotein with a content of 59-67% N-linked carbohydrates and an estimated molecular mass of 160-200 kDa. The extent level of glycosylation affects the thermal behaviour of the enzyme but not its hydrolase and transferase activities, which are optimal at 60-70 °C. The neo-FOS yield of this enzyme was increased from 40 to 168 g/L when the sucrose concentration increased from 420 to 600 g/L and when the reaction was carried out at 60 °C. The neo-FOS levels obtained (168 g/L) in this work are the largest reported for any microbial β-fructofuranosidase.

  8. Extracellular Vesicles, a Key Mediator to Link Environmental Microbiota to Airway Immunity.

    Science.gov (United States)

    Choi, Youngwoo; Park, Hanki; Park, Hae Sim; Kim, Yoon Keun

    2017-03-01

    Asthma is considered the hallmark of chronic airway inflammation, in which several inflammatory cells of the innate and adaptive immune system act together. The disease is thought to be caused by a combination of genetic and environmental factors; however, precise mechanisms for airway inflammation remain unclear. The human microbiota provides an increasingly favored explanation for inflammatory diseases; an altered microbiota composition has been shown to regulate immune responses. However, given the complexity of the microbiota, additional research is needed to elucidate its role in the development of disease. One of the candidate molecules that link microbiota to disease is the extracellular vesicles (EVs). EVs are secreted by diverse cell types and they possess the pathophysiological function of delivering signals between bacteria and host. We discuss the role of the microbiota in the development of asthma through releasing EVs.

  9. Extracellular Saccharide-Mediated Reduction of Au(3+) to Gold Nanoparticles: New Insights for Heavy Metals Biomineralization on Microbial Surfaces.

    Science.gov (United States)

    Kang, Fuxing; Qu, Xiaolei; Alvarez, Pedro J J; Zhu, Dongqiang

    2017-02-15

    Biomineralization is a critical process controlling the biogeochemical cycling, fate, and potential environmental impacts of heavy metals. Despite the indispensability of extracellular polymeric substances (EPS) to microbial life and their ubiquity in soil and aquatic environments, the role played by EPS in the transformation and biomineralization of heavy metals is not well understood. Here, we used gold ion (Au(3+)) as a model heavy metal ion to quantitatively assess the role of EPS in biomineralization and discern the responsible functional groups. Integrated spectroscopic analyses showed that Au(3+)was readily reduced to zerovalent gold nanoparticles (AuNPs, 2-15 nm in size) in aqueous suspension of Escherichia coli or dissolved EPS extracted from microbes. The majority of AuNPs (95.2%) was formed outside Escherichia coli cells, and the removal of EPS attached to cells pronouncedly suppressed Au(3+) reduction, reflecting the predominance of the extracellular matrix in Au(3+) reduction. XPS, UV-vis, and FTIR analyses corroborated that Au(3+) reduction was mediated by the hemiacetal groups (aldehyde equivalents) of reducing saccharides of EPS. Consistently, the kinetics of AuNP formation obeyed pseudo-second-order reaction kinetics with respect to the concentrations of Au(3+) and the hemiacetal groups in EPS, with minimal dependency on the source of microbial EPS. Our findings indicate a previously overlooked, universally significant contribution of EPS to the reduction, mineralization, and potential detoxification of metal species with high oxidation state.

  10. Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer

    Science.gov (United States)

    Soekmadji, Carolina; Riches, James D.; Russell, Pamela J.; Ruelcke, Jayde E.; McPherson, Stephen; Wang, Chenwei; Hovens, Chris M.; Corcoran, Niall M.; Hill, Michelle M.; Nelson, Colleen C.

    2017-01-01

    Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC–MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. PMID:28881726

  11. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and

  12. Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation

    Directory of Open Access Journals (Sweden)

    Kerry L. Inder

    2014-06-01

    Full Text Available Background: Tumour-derived extracellular vesicles (EVs play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis. Methods: We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs. Results: PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikingly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice. Discussion: Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing

  13. Active vitamin D deficiency mediated by extracellular calcium and phosphorus results in male infertility in young mice.

    Science.gov (United States)

    Sun, Weiwei; Chen, Lulu; Zhang, Wei; Wang, Rong; Goltzman, David; Miao, Dengshun

    2015-01-01

    We used mice with targeted deletion of 25-hydroxyvitamin D-1 α-hydroxylase [1α(OH)ase(-/-)] to investigate whether 1,25(OH)2D3 deficiency results in male infertility mediated by 1,25(OH)2D3 or extracellular calcium and phosphorus. Male 1α(OH)ase(-/-) and their wild-type littermates fed either a normal diet or a rescue diet from weaning were mated at 6-14 wk of age with female wild-type mice on the same diet. The fertility efficiency of females was analyzed, and the reproductive phenotypes of males were evaluated by histopathological and molecular techniques. Hypocalcemic and hypophosphatemic male 1α(OH)ase(-/-) mice on a normal diet developed infertility characterized by hypergonadotropic hypogonadism, with downregulation of testicular calcium channels, lower intracellular calcium levels, decreased sperm count and motility, and histological abnormalities of the testes. The proliferation of spermatogenic cells was decreased with downregulation of cyclin E and CDK2 and upregulation of p53 and p21 expression, whereas apoptosis of spermatogenic cells was increased with upregulation of Bax and p-caspase 3 expression and downregulation of Bcl-xl expression. When serum calcium and phosphorus were normalized by the rescue diet, the defective reproductive phenotype in the male 1α(OH)ase(-/-) mice, including the hypergonadotropic hypogonadism, decreased sperm count and motility, histological abnormalities of testis, and defective spermatogenesis, was reversed. These results indicate that the infertility seen in male 1,25(OH)2D3-deficient mice is not a direct effect of active vitamin D deficiency on the reproductive system but is an indirect effect mediated by extracellular calcium and phosphorus. Copyright © 2015 the American Physiological Society.

  14. 重组人血小板源性生长因子增加细胞外信号调节激酶磷酸化促进糖尿病大鼠全层皮肤缺损创面愈合%Recombinant Human Platelet-derived Growth Factor Enhances Repair of Cutaneous Full-thickness Excision by Increasing the Phosphorylation of Extracellular Signal-regulated Kinase in Diabetic Rat

    Institute of Scientific and Technical Information of China (English)

    程飚; 刘宏伟; 付小兵; 盛志勇; 孙同柱

    2006-01-01

    目的 在体观察重组人血小板源性生长因子(recombmant human platelet-derived growth factor,rhPDGF)促进糖尿病大鼠全层皮肤缺损创面修复可能涉及的细胞和分子机制,研究其可能涉及的信号通路.方法 26只糖尿病大鼠,每只动物背部制备4个全层皮肤缺损创面,选取其中52个创面,随机分成3组,即对照组,创面自然愈合;rhPDGF治疗组,创面rhPDGF用量为7.0μg/cm2;赋形剂组,创面用等量赋形剂凝胶.观察治疗后3、7和14 d创面肉芽形成、胶原沉积、再上皮化速率以及炎性细胞浸润情况,并采用免疫荧光和免疫组织化学技术观察创面周围和创面修复细胞内细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)磷酸化和增殖细胞核抗原(proliferative cell nuclear antigen,PCNA)的表达.结果 组织学观察,rhPDGF治疗组创面可见大量炎性细胞浸润,毛细血管胚芽及成纤维细胞明显多于另两组(P<0.05);胶原沉积明显,肉芽组织生长活跃,创面收缩显著,与对照组比较差异有统计学意义(P<0.05).免疫学研究显示,应用rhPDGF 7~14 d后,rhPDGF治疗组ERK1/2明显强于对照组和赋形剂组(P<0.05);且损伤后3~7 d rhPDGF治疗组修复细胞PCNA的表达明显高于对照组和赋形剂组(P<0.05).结论 rhPDGF促糖尿病大鼠创面愈合的作用部分是通过ERK1/2信号通路的磷酸化来完成的.%Objective To investigate the possible signaling mechanisms by which recombinant human platelet-derived growth factor (rhPDGF) accelerated healing of cutaneous wound in diabetic rats. Methods Four full-thickness skin wounds were incised in the back of 26 male Wistar diabetic rats. The wounded rats were divided into 3 groups (7 or 8 rats each group). One group without treatment was used as a control, and the other 2 groups were treated with rhPDGF at a dose of 7.0 μg/cm2 wound or vehicle ( DMSO/0.9%NaCl, vol/vol 1:1) from 1 to 14 days. The wound healing

  15. Heart Rate and Extracellular Sodium and Potassium Modulation of Gap Junction Mediated Conduction in Guinea Pigs.

    Science.gov (United States)

    Entz, Michael; George, Sharon A; Zeitz, Michael J; Raisch, Tristan; Smyth, James W; Poelzing, Steven

    2016-01-01

    Recent studies suggested that cardiac conduction in murine hearts with narrow perinexi and 50% reduced connexin43 (Cx43) expression is more sensitive to relatively physiological changes of extracellular potassium ([K(+)]o) and sodium ([Na(+)]o). Determine whether similar [K(+)]o and [Na(+)]o changes alter conduction velocity (CV) sensitivity to pharmacologic gap junction (GJ) uncoupling in guinea pigs. [K(+)]o and [Na(+)]o were varied in Langendorff perfused guinea pig ventricles (Solution A: [K(+)]o = 4.56 and [Na(+)]o = 153.3 mM. Solution B: [K(+)]o = 6.95 and [Na(+)]o = 145.5 mM). Gap junctions were inhibited with carbenoxolone (CBX) (15 and 30 μM). Epicardial CV was quantified by optical mapping. Perinexal width was measured with transmission electron microscopy. Total and phosphorylated Cx43 were evaluated by western blotting. Solution composition did not alter CV under control conditions or with 15μM CBX. Decreasing the basic cycle length (BCL) of pacing from 300 to 160 ms decreased CV uniformly with both solutions. At 30 μM CBX, a change in solution did not alter CV either longitudinally or transversely at BCL = 300 ms. However, reducing BCL to 160 ms caused CV to decrease more in hearts perfused with Solution B than A. Solution composition did not alter perinexal width, nor did it change total or phosphorylated serine 368 Cx43 expression. These data suggest that the solution dependent CV changes were independent of altered perinexal width or GJ coupling. Action potential duration was always shorter in hearts perfused with Solution B than A, independent of pacing rate and/or CBX concentration. Increased heart rate and GJ uncoupling can unmask small CV differences caused by changing [K(+)]o and [Na(+)]o. These data suggest that modulating extracellular ionic composition may be a novel anti-arrhythmic target in diseases with abnormal GJ coupling, particularly when heart rate cannot be controlled.

  16. Heart Rate and Extracellular Sodium and Potassium Modulation of Gap Junction Mediated Conduction in Guinea Pigs

    Directory of Open Access Journals (Sweden)

    Michael eEntz

    2016-02-01

    Full Text Available Background: Recent studies suggested that cardiac conduction in murine hearts with narrow perinexi and 50% reduced connexin43 (Cx43 expression is more sensitive to relatively physiological changes of extracellular potassium ([K+]o and sodium ([Na+]o. Purpose: Determine whether similar [K+]o and [Na+]o changes alter conduction velocity (CV sensitivity to pharmacologic gap junction (GJ uncoupling in guinea pigs.Methods: [K+]o and [Na+]o were varied in Langendorff perfused guinea pig ventricles (Solution A: [K+]o=4.56 and [Na+]o=153.3 mM. Solution B: [K+]o=6.95 and [Na+]o=145.5 mM. Gap junctions were inhibited with carbenoxolone (CBX (15 and 30 μM. Epicardial CV was quantified by optical mapping. Perinexal width was measured with transmission electron microscopy. Total and phosphorylated Cx43 were evaluated by western blotting. Results: Solution composition did not alter CV under control conditions or with 15M CBX. Decreasing the basic cycle length (BCL of pacing from 300 to 160ms decreased CV uniformly with both solutions. At 30 M CBX, a change in solution did not alter CV either longitudinally or transversely at BCL=300ms. However, reducing BCL to 160ms caused CV to decrease more in hearts perfused with Solution B than A. Solution composition did not alter perinexal width, nor did it change total or phosphorylated serine 368 Cx43 expression. These data suggest that the solution dependent CV changes were independent of altered perinexal width or GJ coupling. Action potential duration was always shorter in hearts perfused with Solution B than A, independent of pacing rate and/or CBX concentration. Conclusions: Increased heart rate and GJ uncoupling can unmask small CV differences caused by changing [K+]o and [Na+]o. These data suggest that modulating extracellular ionic composition may be a novel anti-arrhythmic target in diseases with abnormal GJ coupling, particularly when heart rate cannot be controlled.

  17. Troglitazone induced cytosolic acidification via extracellular signal-response kinase activation and mitochondrial depolarization: complex I proton pumping regulates ammoniagenesis in proximal tubule-like LLC-PK1 cells.

    Science.gov (United States)

    Oliver, Robert; Friday, Ellen; Turturro, Francesco; Welbourne, Tomas

    2008-01-01

    To determined the mechanism(s) through which troglitazone induces cytosolic acidification and glutamine-dependent ammoniagenesis in pig kidney derived LLC-PK1 cells. Acute experiments measured acid extrusion, acid production and simultaneous Extracellular Signal-Regulated Kinase activation. TRO-enhanced acid production was correlated with mitochondrial membrane potential and rotenone and 5-(N-ethyl-N-isopropyl) amiloride, were employed to test specifically the role of Complex I proton pumping. Chronic experiments correlated inhibitors of Complex I with prevention of TRO-increased ammoniagenesis and affects on glutamine metabolism. Exposure to TRO acutely activated Extracellular Signal-Regulated Kinase in a dose dependent manner associated with a fall in spontaneous cytosolic pH. Cytosolic acidosis was associated with both an increase in acid production and inhibition of sodium/hydrogen ion exchanger -mediated acid extrusion. Preventing TRO-induced Extracellular Signal-Regulated Kinase activation with Mitogen Activated Protein Kinase Kinase inhibitors blocked the increase in acid production, restored sodium/hydrogen ion exchanger-activity and prevented cytosolic acidification. Mechanistically, increased acid production was associated with a rapid mitochondrial depolarization and Complex I proton pumping. Blocking Extracellular Signal-Regulated Kinase activation prevented both the fall in Psim and the increased acid production suggesting that the former underlies the accelerated mitochondrial 'acid production'. Mitochondrial Complex I inhibitors EIPA and rotenone prevented increased acid production despite Extracellular Response Kinase activation and reduced sodium/hydrogen ion activity. Inhibition of Complex I prevented TRO's effects on glutamine metabolism. TRO induces cellular acidosis through Extracellular Signal-Regulated Kinase activation-associated acid production and impaired acid extrusion. Acutely, increased acid production reflects mitochondrial Complex I

  18. Extracellular Vesicle-Mediated Reversal of Paclitaxel Resistance in Prostate Cancer

    Science.gov (United States)

    Wang, Justin Q.; DeChalus, Austin; Chatterjee, Devin N.; Keller, Evan T.; Mizokami, Atsushi; Camussi, Giovanni; Mendelsohn, Andrew R.; Renzulli, Joseph F.; Quesenberry, Peter J.; Chatterjee, Devasis

    2017-01-01

    Prostate cancer (PCa) is the most common solid tumor in males and the second leading cause of cancer-related deaths in males in the United States. The current first line therapy for metastatic PCa is androgen deprivation therapy and is initially effective against the disease. However, castrate resistant prostate cancer (CRPC) develops in many men within 18–36 months, rendering this treatment ineffective. Chemotherapy, with a class of drugs known as taxanes is the standard-of-care cytotoxic option in metastatic castrate resistant PCa (mCRPC). However, the overall survival advantage for chemotherapy in mCRPC is only 2.2 months and the cancer cells often become resistant to these drugs as well. Once patients fail chemotherapy the progression to death is inevitable. Extracellular vesicles (EVs) are involved in cell signaling and play a role in cancer progression. Previous work has demonstrated that EVs are involved in the development of drug resistance in cancer cells. We report the reversal of taxane resistance and tumorigenic phenotype in PCa cells after EVs treatment. This study suggests that EVs represent a potentially novel therapeutic treatment option for CRPC. PMID:27279238

  19. Plant-mediated effects on extracellular enzyme activities in distinct soil aggregate size classes in field

    Science.gov (United States)

    Kumar, Amit; Dorodnikov, Maxim; Splettstößer, Thomas; Kuzyakov, Yakov; Pausch, Johanna

    2017-04-01

    Soil aggregation and microbial activities within the aggregates are important factors regulating soil carbon (C) turnover. A reliable and sensitive proxy for microbial activity is activity of extracellular enzymes (EEA). In the present study, effects of soil aggregates on EEA were investigated under three maize plant densities (Low, Normal, and High). Bulk soil was fractionated into three aggregate size classes (>2000 µm large macroaggregates; 2000-250 µm small macroaggregates; moisture sieving. Microbial biomass and EEA (β-1,4-glucosidase (BG), β-1,4-N-acetylglucosaminidase (NAG), L-leucine aminopeptidase (LAP) and acid phosphatase (acP)) catalyzing soil organic matter (SOM) decomposition were measured in rooted soil of maize and soil from bare fallow. Microbial biomass C (Cmic) decreased with decreasing aggregate size classes. Potential and specific EEA (per unit of Cmic) increased from macro- to microaggregates. In comparison with bare fallow soil, specific EEA of microaggregates in rooted soil was higher by up to 73%, 31%, 26%, and 92% for BG, NAG, acP and LAP, respectively. Moreover, high plant density decreased macroaggregates by 9% compared to bare fallow. Enhanced EEA in three aggregate size classes demonstrated activation of microorganisms by roots. Strong EEA in microaggregates can be explained by microaggregates' localization within the soil. Originally adhering to surfaces of macroaggregates, microaggregates were preferentially exposed to C substrates and nutrients, thereby promoting microbial activity.

  20. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    Energy Technology Data Exchange (ETDEWEB)

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Neto Paiva, Claudia; Torres Bozza, Marcelo [Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Rosado Fantappie, Marcelo, E-mail: fantappie@bioqmed.ufrj.br [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil)

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  1. Alterations in junctional proteins, inflammatory mediators and extracellular matrix molecules in eosinophilic esophagitis.

    Science.gov (United States)

    Abdulnour-Nakhoul, Solange M; Al-Tawil, Youhanna; Gyftopoulos, Alex A; Brown, Karen L; Hansen, Molly; Butcher, Kathy F; Eidelwein, Alexandra P; Noel, Robert A; Rabon, Edd; Posta, Allison; Nakhoul, Nazih L

    2013-08-01

    Eosinophilic esophagitis (EoE), an inflammatory atopic disease of the esophagus, causes massive eosinophil infiltration, basal cell hyperplasia, and sub-epithelial fibrosis. To elucidate cellular and molecular factors involved in esophageal tissue damage and remodeling, we examined pinch biopsies from EoE and normal pediatric patients. An inflammation gene array confirmed that eotaxin-3, its receptor CCR3 and interleukins IL-13 and IL-5 were upregulated. An extracellular matrix (ECM) gene array revealed upregulation of CD44 & CD54, and of ECM proteases (ADAMTS1 & MMP14). A cytokine antibody array showed a marked decrease in IL-1α and IL-1 receptor antagonist and an increase in eotaxin-2 and epidermal growth factor. Western analysis indicated reduced expression of intercellular junction proteins, E-cadherin and claudin-1 and increased expression of occludin and vimentin. We have identified a number of novel genes and proteins whose expression is altered in EoE. These findings provide new insights into the molecular mechanisms of the disease.

  2. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    Science.gov (United States)

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

  3. Effect of extracellular signal regulated kinase on cultured hippocampal neurons of rats with oxygen-g1ucose deprivation%细胞外信号调节激酶对氧糖剥夺后大鼠海马神经元的作用研究

    Institute of Scientific and Technical Information of China (English)

    华烨; 丁新生; 孔岳南

    2011-01-01

    Objective: To investigate the effect of excelluar signal-regulated kinase (ERK) on rat hippocampal neurons with oxygen-glucose deprivation (OGD). Methods:Cultured hippocampal neurons were designed into four groups:normal group,OGD group,PD98059 10 μmol/L and 30 μmoL/L groups. The apoptosis of neurons was determined by flow cytometry, and Western blot was used to evaluate protein expression of ERKI/2 and pERKI/2. Results:Compared with normal control group, neuron apoptosis increased while pERK1/2 protein expression decreased obviously in OGD group (both P < 0.01). Neuron apoptosis increased and pERK1/2 protein expression decreased more significantly in PD98059 groups compared with OGD group (both P < 0.01 ). Changes in 30 μmol/L PD98059 group were more significant than 10 μmol/L PD98059 group (P < 0.01 ). Each group showed no significant change in ERK expression (P > 0.05). Conclusion:ERK may be involved in neuronal apoptosis after OGD, and inhibition of ERK pathway can promote neuronal apoptosis.%目的:研究细胞外信号调节激酶对氧糖剥夺(oxygen-glucose deprivation,OGD)后大鼠海马神经元的作用.方法:建立培养乳鼠海马神经元OGD模型,并分为正常对照组、OGD组、PD 98059 10 μmol/L、30 μmol/L组.流式细胞仪Annexin V/PI双染色法检测神经元细胞凋亡率,Western blot法检测ERKl/2、pERKl/2的表达.结果:与正常对照组相比,OGD组神经元的凋亡率升高(P<0.01),pERKl/2的表达降低(P<0.01),与OGD组相比,PD98059组神经元凋亡率明显升高(P<0.01),pERKl/2的表达明显降低(P<0.01),30;μmol/L组较10 μmol/L组凋亡率升高及pERKl/2表达降低更为显著(P<0.01),各组ERK含量无明显变化(P>0.05).结论:ERK可能参与氧糖剥夺后的神经元凋亡,抑制ERK通路可促进神经元凋亡.

  4. Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Holm, Rikke; Vilsen, Bente;

    2016-01-01

    , in addition, Na+/K+-ATPase-mediated K+ clearance could be governed by astrocytic [Na+]i. During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+-coupled glutamate transporters, thereby elevating [Na+]i. It thus remains unresolved whether the different Na...... constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+/K+-ATPase was not fully saturated at basal astrocytic [Na+]i, irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2...

  5. Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

    Science.gov (United States)

    Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

    2008-07-01

    Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.

  6. Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe

    2007-01-01

    The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process...... in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular...... collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis...

  7. Podoplanin-mediated cell adhesion through extracellular matrix in oral squamous cell carcinoma.

    Science.gov (United States)

    Tsuneki, Masayuki; Yamazaki, Manabu; Maruyama, Satoshi; Cheng, Jun; Saku, Takashi

    2013-08-01

    Podoplanin (PDPN), one of the representative mucin-like type-I transmembrane glycoproteins specific to lymphatic endothelial cells, is expressed in various cancers including squamous cell carcinoma (SCC). On the basis of our previous studies, we have developed the hypothesis that PDPN functions in association with the extracellular matrix (ECM) from the cell surface side. The aim of this study was to elucidate the molecular role of PDPN in terms of cell adhesion, proliferation, and migration in oral SCC cells. Forty-four surgical specimens of oral SCC were used for immunohistochemistry for PDPN, and the expression profiles were correlated with their clinicopathological properties. Using ZK-1, a human oral SCC cell system, and five other cell systems, we examined PDPN expression levels by immunofluorescence, western blotting, and real-time PCR. The effects of transient PDPN knockdown by siRNA in ZK-1 were determined for cellular functions in terms of cell proliferation, adhesion, migration, and invasion in association with CD44 and hyaluronan. Cases without PDPN-positive cells were histopathologically classified as less-differentiated SCC, and SCC cells without PDPN more frequently invaded lymphatics. Adhesive properties of ZK-1 were significantly inhibited by siRNA, and PDPN was shown to collaborate with CD44 in cell adhesion to tether SCC cells with hyaluronan-rich ECM of the narrow intercellular space as well as with the stromal ECM. There was no siRNA effect in migration. We have demonstrated the primary function of PDPN in cell adhesion to ECM, which is to secondarily promote oral SCC cell proliferation.

  8. Activin Receptor Signaling Regulates Prostatic Epithelial Cell Adhesion and Viability

    Directory of Open Access Journals (Sweden)

    Derek P. Simon

    2009-04-01

    Full Text Available Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s in cell proliferation. Using prostatic cancer cell (PCC lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII, as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS. Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

  9. Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles.

    Science.gov (United States)

    Giricz, Zoltán; Varga, Zoltán V; Baranyai, Tamás; Sipos, Péter; Pálóczi, Krisztina; Kittel, Ágnes; Buzás, Edit I; Ferdinandy, Péter

    2014-03-01

    Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100 nm and 100-1000 nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, renders EV ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3 × 5-5 min global ischemia and reperfusion (IPC) or 30 min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30 min global ischemia and 120 min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9 ± 1.6% vs. 25.0 ± 2.7%), similarly to cardioprotection afforded by IPC (7.3 ± 2.7% vs. 22.1 ± 2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0 ± 2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular

  10. Upregulation of parathyroid VDR expression by extracellular calcium is mediated by ERK1/2-MAPK signaling pathway.

    Science.gov (United States)

    Cañadillas, Sagrario; Canalejo, Rocio; Rodriguez-Ortiz, Maria Encarnacion; Martinez-Moreno, Julio Manuel; Estepa, Jose Carlos; Zafra, Rafael; Perez, Jose; Muñoz-Castañeda, Juan Rafael; Canalejo, Antonio; Rodriguez, Mariano; Almaden, Yolanda

    2010-05-01

    We have previously demonstrated that the activation of rat parathyroid calcium-sensing receptor (CaSR) upregulates VDR expression in vivo (Garfia B, Cañadillas S, Luque F, Siendones E, Quesada M, Almadén Y, Aguilera-Tejero E, Rodríguez M. J Am Soc Nephrol 13: 2945-2952, 2002; Rodriguez ME, Almaden Y, Cañadillas S, Canalejo A, Siendones E, Lopez I, Aguilera-Tejero E, Martin D, Rodriguez M. Am J Physiol Renal Physiol 292: F1390-F1395, 2007). The present study was designed to characterize the signaling system that mediates the stimulation of parathyroid VDR gene expression by extracellular calcium. Experiments were performed in vitro by the incubation of rat parathyroid glands and in vivo with normal and uremic (Nx) rats receiving injections of CaCl(2) or EDTA to obtain hypercalcemic or hypocalcemic clamps. A high calcium concentration increased VDR expression. The addition of arachidonic acid (AA) to the low-calcium medium produced an increase in VDR mRNA of the same magnitude as that observed with high calcium. The addition of ionophore to the low-calcium medium also increased VDR mRNA expression. High calcium or the addition of AA to the low-calcium medium induced the activation (phosphorylation) of ERK1/2-MAPK. The specific inhibition of the ERK1/2-MAPK activity prevented the stimulation of VDR expression by high calcium or AA. These results suggest that AA regulates parathyroid VDR gene expression through the activation of the ERK1/2-MAPK. CaSR activation induced the activation of transcription factor Sp1, but not of NF-κB p50 or p65 or activator protein-1. The addition of AA to the low-calcium medium increased specific DNA-binding activity of Sp1 to almost the same level as high calcium, which was prevented by the inhibition of ERK1/2. Furthermore, mithramycin A (a Sp1 inhibitor) prevented the upregulation of VDR mRNA by high calcium. Finally, both sham and Nx hypercalcemic rats showed similar increased levels of VDR mRNA compared with sham and Nx

  11. Glucose- and interleukin-1beta-induced beta-cell apoptosis requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets

    DEFF Research Database (Denmark)

    Maedler, Kathrin; Størling, Joachim; Sturis, Jeppe

    2004-01-01

    Increasing evidence indicates that a progressive decrease in the functional beta-cell mass is the hallmark of both type 1 and type 2 diabetes. The underlying causes, beta-cell apoptosis and impaired secretory function, seem to be partly mediated by macrophage production of interleukin (IL)-1beta......-regulated kinase (ERK) 1/2, an effect that was abrogated by 3 micromol/l NN414. Similarly, 1 micromol/l of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor PD098059 or 1 micromol/l of the l-type Ca(2+) channel blocker nimodipine prevented glucose- and IL-1beta-induced ERK activation, beta...

  12. 2,4,6-Trichlorophenol mediated increases in extracellular peroxidase activity in three species of Lemnaceae.

    Science.gov (United States)

    Biswas, Dilip K; Scannell, Gillian; Akhmetov, Nurlan; Fitzpatrick, Dara; Jansen, Marcel A K

    2010-11-01

    Chlorinated phenols, or chlorophenols, are persistent priority pollutants that are widespread in the environment. Class III peroxidases are well-characterised plant enzymes that can catalyse the oxidative dechlorination of chlorophenols. Expression of these enzymes by plants is commonly associated with plant stress, therefore limiting scope for phytoremediation. In this study, we have quantitatively compared peroxidase activity and phytotoxicity as a function of 2,4,6-trichlorophenol (TCP) concentration in three species of Lemnaceae; Lemna minor, Lemna gibba and Landoltia punctata. Effects of TCP on the growth rates of the three species differed considerably with L. punctata being the most tolerant species. TCP also affected photosynthetic parameters, causing a decrease in open photosystem II reaction centres (qP) and, in L. punctata only, a decrease in non-photochemical quenching (qN). In parallel, TCP exposure resulted in increased peroxidase activity in all three species. Peroxidase activity in L. minor and L. gibba displayed an inverse relationship with biomass accumulation, i.e. the more growth reduction the more peroxidase activity. In contrast, induction of peroxidase activity in L. punctata was bi-phasic, with a TCP-induced activity peak at concentrations that had no major effect on growth, and further induction under phytotoxic concentrations. The mechanism by which L. punctata recognises and responds to low concentrations of an anthropogenic compound, in the absence of wide-ranging stress, remains enigmatic. However, we conclude that this "window" of peroxidase production in the absence of major growth inhibition offers potential for the development of sustainable, peroxidise-mediated phytoremediation systems. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. CD11b/CD18 (Mac-1) is a novel surface receptor for extracellular double-stranded RNA to mediate cellular inflammatory responses.

    Science.gov (United States)

    Zhou, Hui; Liao, Jieying; Aloor, Jim; Nie, Hui; Wilson, Belinda C; Fessler, Michael B; Gao, Hui-Ming; Hong, Jau-Shyong

    2013-01-01

    During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1 [macrophage-1 Ag]), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.

  14. Direct excitation of inhibitory interneurons by extracellular ATP mediated by P2Y1 receptors in the hippocampal slice.

    Science.gov (United States)

    Kawamura, Masahito; Gachet, Christian; Inoue, Kazuhide; Kato, Fusao

    2004-12-01

    ATP is an important cell-to-cell signaling molecule mediating the interactions between astrocytes and neurons in the CNS. In the hippocampal slices, ATP suppresses excitatory transmission mostly through activation of adenosine A1 receptors, because the ectoenzyme activity for the extracellular breakdown of ATP to adenosine is high in slice preparations in contrast to culture environments. Because the hippocampus is also rich in the expression of P2 receptors activated specifically by ATP, we examined whether ATP modulates neuronal excitability in the acute slice preparations independently of adenosine receptors. Although ATP decreased the frequency of spontaneously occurring EPSCs in the CA3 pyramidal neurons through activation of adenosine A1 receptors, ATP concurrently increased the frequency of IPSCs in a manner dependent on action potential generation. This effect was mediated by P2Y1 receptors because (1) 2-methylthio-ATP (2meSATP) was the most potent agonist, (2) 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate diammonium (MRS2179) abolished this effect, and (3) this increase in IPSC frequency was not observed in the transgenic mice lacking P2Y1 receptor proteins. Application of 2meSATP elicited MRS2179-sensitive time- and voltage-dependent inward currents in the interneurons, which depolarized the cell to firing threshold. Also, it increased [Ca2+]i in both astrocytes and interneurons, but, unlike the former effect, the latter was entirely dependent on Ca2+ entry. Thus, in hippocampal slices, in addition to activating A1 receptors of the excitatory terminals after being converted to adenosine, ATP activates P2Y1 receptors in the interneurons, which is linked to activation of unidentified excitatory conductance, through mechanisms distinct from those in the astrocytes.

  15. Glucose- and interleukin-1beta-induced beta-cell apoptosis requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets

    DEFF Research Database (Denmark)

    Maedler, Kathrin; Størling, Joachim; Sturis, Jeppe

    2004-01-01

    -regulated kinase (ERK) 1/2, an effect that was abrogated by 3 micromol/l NN414. Similarly, 1 micromol/l of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor PD098059 or 1 micromol/l of the l-type Ca(2+) channel blocker nimodipine prevented glucose- and IL-1beta-induced ERK activation, beta......Increasing evidence indicates that a progressive decrease in the functional beta-cell mass is the hallmark of both type 1 and type 2 diabetes. The underlying causes, beta-cell apoptosis and impaired secretory function, seem to be partly mediated by macrophage production of interleukin (IL)-1beta...... and/or high-glucose-induced beta-cell production of IL-1beta. Treatment of type 1 and type 2 diabetic patients with the potassium channel opener diazoxide partially restores insulin secretion. Therefore, we studied the effect of diazoxide and of the novel potassium channel opener NN414, selective...

  16. Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

    Directory of Open Access Journals (Sweden)

    Kirsten Ridder

    2014-06-01

    Full Text Available Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

  17. Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

    Science.gov (United States)

    Murmann, Tamara; Carrillo-García, Carmen; Veit, Nadine; Courts, Cornelius; Glassmann, Alexander; Janzen, Viktor; Madea, Burkhard; Reinartz, Markus; Harzen, Anne; Nowak, Michael; Perner, Sven; Winter, Jochen; Probstmeier, Rainer

    2014-01-01

    Small cell lung carcinomas (SCLCs) represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

  18. Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

    Directory of Open Access Journals (Sweden)

    Tamara Murmann

    Full Text Available Small cell lung carcinomas (SCLCs represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

  19. Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition.

    OpenAIRE

    Schreiber, M; Sedger, L; McFadden, G

    1997-01-01

    The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T...

  20. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    Energy Technology Data Exchange (ETDEWEB)

    McCready, Jessica [Department of Natural Sciences, Assumption College, Worcester, MA 01609 (United States); Wong, Daniel S. [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States); Burlison, Joseph A.; Ying, Weiwen [Synta Pharmaceuticals, Lexington, MA 02421 (United States); Jay, Daniel G., E-mail: daniel.jay@tufts.edu [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States)

    2014-04-30

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

  1. Canonical Wnt signalling regulates epithelial patterning by modulating levels of laminins in zebrafish appendages.

    Science.gov (United States)

    Nagendran, Monica; Arora, Prateek; Gori, Payal; Mulay, Aditya; Ray, Shinjini; Jacob, Tressa; Sonawane, Mahendra

    2015-01-15

    The patterning and morphogenesis of body appendages - such as limbs and fins - is orchestrated by the activities of several developmental pathways. Wnt signalling is essential for the induction of limbs. However, it is unclear whether a canonical Wnt signalling gradient exists and regulates the patterning of epithelium in vertebrate appendages. Using an evolutionarily old appendage - the median fin in zebrafish - as a model, we show that the fin epithelium exhibits graded changes in cellular morphology along the proximo-distal axis. This epithelial pattern is strictly correlated with the gradient of canonical Wnt signalling activity. By combining genetic analyses with cellular imaging, we show that canonical Wnt signalling regulates epithelial cell morphology by modulating the levels of laminins, which are extracellular matrix components. We have unravelled a hitherto unknown mechanism involved in epithelial patterning, which is also conserved in the pectoral fins - evolutionarily recent appendages that are homologous to tetrapod limbs.

  2. Involvement of Extracellular Signal Regulated Kinases in Traumatic Brain Injury-Induced Depression in Rodents

    OpenAIRE

    Kuo, Jinn-Rung; Cheng, Yi-Hsuan; Chen, Yi-Shion; Chio, Chung-Ching; Gean, Po-Wu

    2013-01-01

    Traumatic brain injury (TBI) is the most common cause of death and acquired disability among children and young adults in the developed countries. In clinical studies, the incidence of depression is high after TBI, and the mechanisms behind TBI-induced depression remain unclear. In the present study, we subjected rats to a moderate fluid percussion into the closed cranial cavity to induce TBI. After 3 days of recovery, injured rats were given a forced swim test (FST) and novelty-suppressed fe...

  3. Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Strom, Christina N; Bailey, Sean T

    2008-01-01

    Leptin was initially identified as a regulator of appetite and weight control centers in the hypothalamus, but appears to be involved in a number of physiological processes. This study was carried out to examine the possible role of leptin in regulating prolactin (PRL) release using the teleost...... pituitary model system. This advantageous system allows isolation of a nearly pure population of lactotropes in their natural, in situ aggregated state. The rostral pars distalis were dissected from tilapia pituitaries and exposed to varying concentrations of leptin (0, 1, 10, 100 nM) for 1 h. Release...... of PRL was stimulated by leptin in a potent and concentration-dependent manner. A time-course experiment showed that the strongest response in PRL release with leptin occurs within the first hour (approximately sixfold), and stimulation was sustained after 16 h (approximately twofold). Many...

  4. 鞘内注射γ-氨基丁酸转运体抑制剂NO-711在骨癌痛大鼠脊髓水平印制磷酸化细胞外信号周节激酶1/2的上调%Intrathecal administration of γ-aminobutyric acid transporter-1 inhibitor NO-711 inhibits the up-regulation of extracellular signal-regulated kinase in lumbar spinal cord of bone cancer pain in rats

    Institute of Scientific and Technical Information of China (English)

    朱珊珊; 谭珊珊; 曾因明

    2011-01-01

    Objective To explore the role of extracellular signal-regulated kirase(ERK)activation in the lumbar spinal cord induced by the tibial cancer pain in rats.Methods Part Ⅰ:Forty-eight SD female rats(160 g-200 g)were randomly divided into two groups(n=24).Group A(control group):intra-tibial injection of 5 p.l saline.Group B(model group):intra-tibial injection of 5 μl Walker-256 mammary gland carcinoma cells of rats(1×105/μl).Mechanical withdrawl threshold(MWT)of mechanical stimulus and ambulatory-evoked pain scores were recorded one day before operation and on 1,3,5,7,10,14,21 d after operation respectively.Expressionof phosphorylated ERK1/2(p-ERK1/2)were detected on 7,14,21 d after operation respectively.Part Ⅱ:Sixty female Sprague-Dawley rats(160 g-200 g)were randomly divided into five groups(n=12):sham +NS group(group S);bone cancer pain+NS(group N1);bone cancer pain +NO-711 10 μg(group N2);bone cancer pain+ NO-711 20 μg(group N3);bone cancer pain+ NO-711 40 μg(group N4).On the 14th day after intra-tibial injection of 5 μ.l Walker-256 mammary gland carcinoma cells(post-treatment)NO-711 or normal saline were intrathecally administered thrice(once a day).MWT of mechanical stimulus and ambulatory-evoked pain scores were recorded at the time of 30 min,1,2,4,8,12,24 h after last injection.Test the p-ERK1/2 expression in the spinal cord by western blot analysis at 4 hours after last injection.Results Part Ⅰ:From the 7th day after operation,there were great significance differences at MWT(8.02±0.30)、ambulatory-evoked pain scores(0.88±0.22)and the expression of p-ERK1/2 compared with pre-operation in group B.MWT were significantly decreased while ambulatory-evoked pain scores and the expression of p-ERK1/2 were significanty increased in group B as compared to group A from the 7th day postoperation.Part Ⅱ:After and before i.t.administration in group N 1 the MWT and scores of ambulatory-evoked pain showed no statistical significance.Received with

  5. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

    Science.gov (United States)

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

  6. Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

    Directory of Open Access Journals (Sweden)

    Guduric-Fuchs Jasenka

    2012-08-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs, and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. Results Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2 is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p. Conclusion The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

  7. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  8. 司坦唑醇激活大鼠生长板软骨细胞雌激素受体α、胰岛素样生长因子1受体和细胞外信号调节激酶1/2的交联对话%Stanozolol activates the cross-talk of estrogen receptor α-insulin-like growth factor-1 receptor-extracellular-signal regulated kinase 1/2 in the growth plate chondrocytes of estrogen-inhibited adolescent rats in vitro

    Institute of Scientific and Technical Information of China (English)

    朱顺叶; 李燕虹; 马华梅; 潘思年; 陈红珊; 杜敏联

    2009-01-01

    Objective To investigate the effects and the mechanisms of stanozolol (ST) on the proliferation, maturation and differentiation of in vitro cultured growth plate chondrocyte isolated from gonadotropin releasing hormone analogue (GnRHa)-treated adolescent rats, to study if ST mediates the proliferation of chondrocytes via the estrogen receptor α (ERα), androgen receptor (AR) and/α insulin-like growth factor-1 receptor (IGF-1R) and interactions of the two receptor and IGF-1R receptor signaling pathway, to investigate the mechanism of the biological effects in ST promoting bone growth/maturity at molecular level. Method The rats were weaned at the end of 3 weeks and intramuscular injection of triptorelin of GnRHa preparations, qow×2 was started. The rats were sacrificed at the end of 7 weeks, and then the tibiae growth plates were taken out with sterile procedure. The chondrocytes were obtained by two-time enzyme digestion method, and the experiments were carried out with the primary chondrocytes. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and Western blot analysis were applied. Result The results of PCNA demonstrated that stanozolol enhanced the proliferation of the chondrocytes, time-course studies showed that the proliferation were maximally stimulated by stanozolol after 2 days of incubation and decreased again after longer periods of incubation. The expression of p-ERα, p-IGF-1 R and p-extracellular-signal regulated kinase 1/2 (ERK1/2) increased with the incubation period of ST treatment, and reached the peak value at a certain time, and then gradually decreased. The expression of p-ERα, p-IGF-1R and p-ERK1/2 increased with the elevation of ST concentration, and reached the peak value at 10~(-9)-10~(-8) mol/L, then gradually decreased. ST induced-p-ERα expression was partially blocked by ERα and mitogen-activated protein kinase kinase inhibitors. ST induced-p-IGF-1R expression was partially blocked by ERα and [GF-1R

  9. Timing of the developmental switch in GABA(A) mediated signaling from excitation to inhibition in CA3 rat hippocampus using gramicidin perforated patch and extracellular recordings.

    Science.gov (United States)

    Tyzio, Roman; Holmes, Gregory L; Ben-Ari, Yehezkiel; Khazipov, Roustem

    2007-01-01

    The timing of the developmental switch in the GABA(A) mediated responses from excitatory to inhibitory was studied in Wistar rat CA3 hippocampal pyramidal cells using gramicidin perforated patch-clamp and extracellular recordings. Gramicidin perforated patch recordings revealed a gradual developmental shift in the reversal potential of synaptic and isoguvacine-induced GABA(A) mediated responses from -55 +/- 4 mV at postnatal days P0-2 to -74 +/- 3 mV at P13-15 with a midpoint of disappearance of the excitatory effects of GABA at around P8. Extracellular recordings in CA3 pyramidal cell layer revealed that the effect of isoguvacine on multiple unit activity (MUA) switched from an increase to a decrease at around P10. The effect of synaptic GABA(A) mediated responses on MUA switched from an increase to a decrease at around P8. It is concluded that the developmental switch in the action of GABA via GABA(A) receptors from excitatory to inhibitory occurs in Wistar rat CA3 pyramidal cells at around P8-10, an age that coincides with the transition from immature to mature hippocampal rhythms. We propose that excitatory GABA contributes to enhanced excitability and ictogenesis in the neonatal rat hippocampus.

  10. The key residue within the second extracellular loop of human EP3 involved in selectively turning down PGE2- and retaining PGE1-mediated signaling in live cells.

    Science.gov (United States)

    Akasaka, Hironari; Thaliachery, Natasha; Zheng, Xianghai; Blumenthal, Marissa; Nikhar, Sameer; Murdoch, Emma E; Ling, Qinglan; Ruan, Ke-He

    2017-02-15

    Key residues and binding mechanisms of PGE1 and PGE2 on prostanoid receptors are poorly understood due to the lack of X-ray structures for the receptors. We constructed a human EP3 (hEP3) model through integrative homology modeling using the X-ray structure of the β2-adrenergic receptor transmembrane domain and NMR structures of the thromboxane A2 receptor extracellular loops. PGE1 and PGE2 docking into the hEP3 model showed differing configurations within the extracellular ligand recognition site. While PGE2 could form possible binding contact with S211, PGE1 is unable to form similar contacts. Therefore, S211 could be the critical residue for PGE2 recognition, but is not a significant for PGE1. This prediction was confirmed using HEK293 cells transfected with hEP3 S211L cDNA. The S211L cells lost PGE2 binding and signaling. Interestingly, the S211L cells retained PGE1-mediated signaling. It indicates that S211 within the second extracellular loop is a key residue involved in turning down PGE2 signaling. Our study provided information that S211L within EP3 is the key residue to distinguish PGE1 and PGE2 binding to mediate diverse biological functions at the initial recognition step. The S211L mutant could be used as a model for studying the binding mechanism and signaling pathway specifically mediated by PGE1. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis.

    Science.gov (United States)

    Bonne-Année, Sandra; Kerepesi, Laura A; Hess, Jessica A; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B; Nolan, Thomas J; Abraham, David

    2014-06-01

    Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.

  12. Drosophila tensin plays an essential role in cell migration and planar polarity formation during oogenesis by mediating integrin-dependent extracellular signals to actin organization.

    Science.gov (United States)

    Cha, In Jun; Lee, Jang Ho; Cho, Kyoung Sang; Lee, Sung Bae

    2017-03-11

    Oogenesis in Drosophila involves very dynamic cellular changes such as cell migration and polarity formation inside an ovary during short period. Previous studies identified a number of membrane-bound receptors directly receiving certain types of extracellular inputs as well as intracellular signalings to be involved in the regulation of these dynamic cellular changes. However, yet our understanding on exactly how these receptor-mediated extracellular inputs lead to dynamic cellular changes remains largely unclear. Here, we identified Drosophila tensin encoded by blistery (by) as a novel regulator of cell migration and planar polarity formation and characterized the genetic interaction between tensin and integrin during oogenesis. Eggs from by mutant showed decreased hatching rate and morphological abnormality, a round-shape, compared to the wild-type eggs. Further analyses revealed that obvious cellular defects such as defective border cell migration and planar polarity formation might be primarily associated with the decreased hatching rate and the round-shape phenotype of by mutant eggs, respectively. Moreover, by mutation also induced marked defects in F-actin organization closely associated with both cell migration and planar polarity formation during oogenesis of Drosophila. Notably, all these defective phenotypes observed in by mutant eggs became much severer by reduced level of integrin, indicative of a close functional association between integrin and tensin during oogenesis. Collectively, our findings suggest that tensin acts as a crucial regulator of dynamic cellular changes during oogenesis by bridging integrin-dependent extracellular signals to intracellular cytoskeletal organization.

  13. Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR.

    Science.gov (United States)

    Young, Robert L; Malcolm, Kenneth C; Kret, Jennifer E; Caceres, Silvia M; Poch, Katie R; Nichols, David P; Taylor-Cousar, Jennifer L; Saavedra, Milene T; Randell, Scott H; Vasil, Michael L; Burns, Jane L; Moskowitz, Samuel M; Nick, Jerry A

    2011-01-01

    The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by

  14. Neutrophil extracellular trap (NET-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR.

    Directory of Open Access Journals (Sweden)

    Robert L Young

    Full Text Available The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs, extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated

  15. Extracellular ATP mediates necrotic cell swelling in SN4741 dopaminergic neurons through P2X7 receptors.

    Science.gov (United States)

    Jun, Dong-Jae; Kim, Jaeyoon; Jung, Sang-Yong; Song, Ran; Noh, Ji-Hyun; Park, Yong-Soo; Ryu, Sung-Ho; Kim, Joung-Hun; Kong, Young-Yun; Chung, Jun-Mo; Kim, Kyong-Tai

    2007-12-28

    Extracellular ATP has recently been identified as an important regulator of cell death in response to pathological insults. When SN4741 cells, which are dopaminergic neurons derived from the substantia nigra of transgenic mouse embryos, are exposed to ATP, cell death occurs. This cell death is associated with prominent cell swelling, loss of ER integrity, the formation of many large cytoplasmic vacuoles, and subsequent cytolysis and DNA release. In addition, the cleavage of caspase-3, a hallmark of apoptosis, is induced by ATP treatment. However, caspase inhibitors do not overcome ATP-induced cell death, indicating that both necrosis and apoptosis are associated with ATP-induced cell death and suggesting that a necrotic event might override the apoptotic process. In this study we also found that P2X(7) receptors (P2X(7)Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X(7)Rs antagonist, KN62, or by knock-down of P2X(7)Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X(7)Rs.

  16. Characterization of a region of the lutropin receptor extracellular domain near transmembrane helix 1 that is important in ligand-mediated signaling.

    Science.gov (United States)

    Alvarez, C A; Narayan, P; Huang, J; Puett, D

    1999-04-01

    The lutropin receptor (LHR), a member of the G protein-coupled receptor family, contains a relatively large N-terminal extracellular domain, accounting for about half of the receptor and responsible for high affinity ligand binding, and a standard heptahelical portion with connecting loops and a C-terminal tail. LHR and the other two glycoprotein hormone receptors, i.e. the follitropin and TSH receptors, contain an invariant 10-amino acid residue sequence, FNPCEDIMGY (residues 328-337 in rat LHR), in the extracellular domain separated by only a few amino acid residues from the beginning of transmembrane helix 1. In view of the invariant nature of this region in the three glycoprotein hormone receptors and preliminary data in the literature on the importance of Glu332 and Asp333 in signal transduction, we undertook a systematic investigation of all 10 amino acid residues because this region may function as a switch or trigger for communicating ligand binding to the extracellular domain with a conformational change of the membrane-embedded C-terminal half of the receptor to activate G proteins, particularly Gs. A total of 36 single, double, and multiple replacements, as well as two deletions, of LHR were prepared and characterized in transiently transfected COS-7 cells. Of these mutants LHRs, 26 expressed on the cell surface in sufficient numbers that quantitative assessments could be made of human choriogonadotropin binding and ligand-mediated cAMP production. Replacements of Cys331 abolished ligand binding to intact cells, although binding could be detected after solubilization of the cells. Replacements of the other nine amino acid residues that did not interfere with receptor folding or trafficking had no significant effect on ligand binding affinity; however, replacements of Pro330, Glu332, and Asp333 resulted in diminished signaling, especially for the two acidic residues. An interesting observation was made in which replacement of Tyr337 with Ala or Asp, while

  17. Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric ModulatorS⃞

    Science.gov (United States)

    Smith, Nicola J.; Ward, Richard J.; Stoddart, Leigh A.; Hudson, Brian D.; Kostenis, Evi; Ulven, Trond; Morris, Joanne C.; Tränkle, Christian; Tikhonova, Irina G.; Adams, David R.

    2011-01-01

    Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB. PMID:21498659

  18. T Cell-Independent Mechanisms Associated with Neutrophil Extracellular Trap Formation and Selective Autophagy in IL-17A-Mediated Epidermal Hyperplasia.

    Science.gov (United States)

    Suzuki, Erika; Maverakis, Emanual; Sarin, Ritu; Bouchareychas, Laura; Kuchroo, Vijay K; Nestle, Frank O; Adamopoulos, Iannis E

    2016-12-01

    IL-17A has been strongly associated with epidermal hyperplasia in many cutaneous disorders. However, because IL-17A is mainly produced by αβ and γδT cells in response to IL-23, the role of T cells and IL-23 has overshadowed any IL-17A-independent actions. In this article, we report that IL-17A gene transfer induces epidermal hyperplasia in Il23r(-/-)Rag1(-/-)- and Tcrδ-deficient mice, which can be prevented by neutrophil depletion. Moreover, adoptive transfer of CD11b(+)Gr-1(hi) cells, after IL-17A gene transfer, was sufficient to phenocopy the disease. We further show that the IL-17A-induced pathology was prevented in transgenic mice with impaired neutrophil extracellular trap formation and/or neutrophils with conditional deletion of the master regulator of selective autophagy, Wdfy3. Our data demonstrate a novel T cell-independent mechanism that is associated with neutrophil extracellular trap formation and selective autophagy in IL-17A-mediated epidermal hyperplasia. Copyright © 2016 by The American Association of Immunologists, Inc.

  19. Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and lck signaling.

    Science.gov (United States)

    Sharp, L L; Hedrick, S M

    1999-12-15

    The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.

  20. The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity.

    Science.gov (United States)

    Yu, Rongjie; Liu, Hongyu; Peng, Xinhe; Cui, Yue; Song, Suqin; Wang, Like; Zhang, Huahua; Hong, An; Zhou, Tianhong

    2017-06-27

    VPAC1 is class B G protein-coupled receptors (GPCR) shared by pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). The first cysteine (Cys37) in the N-terminal extracellular domain of mature VPAC1 is a free Cys not involved in the formation of conserved intramolecular disulfide bonds. In order to investigate the biological role of this Cys37 in VPAC1, the wild-type VPAC1 and Cys37/Ala mutant (VPAC1-C37/A) were expressed stably as fusion proteins with enhanced yellow fluorescent protein (EYFP) respectively in Chinese hamster ovary (CHO) cells. Both VPAC1-EYFP and VPAC1-C37/A-EYFP trafficked to the plasma membrane normally, and CHO cells expressing VPAC1-EYFP displayed higher anti-apoptotic activity against camptothecin (CPT) induced apoptosis than the cells expressing VPAC1-C37/A-EYFP, while VPAC1-C37/A-CHO cells showed higher proliferative activity than VPAC1-CHO cells. Confocal microscopic analysis, western blotting and fluorescence quantification assay showed VPAC1-EYFP displayed significant nuclear translocation while VPAC1-C37/A-EYFP did not transfer into nucleus under the stimulation of VIP (0.1 nM). Acyl-biotin exchange assay and click chemistry-based palmitoylation assay confirmed for the first time the palmitoylation of Cys37, which has been predicted by bioinformatics analysis. And the palmitoylation inhibitor 2-bromopalmitate significantly inhibited the nuclear translocation of VPAC1-EYFP and its anti-apoptotic activity synchronously. These results indicated the palmitoylation of the Cys37 in the N-terminal extracellular domain of VPAC1 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity. These findings reveal for the first time the lipidation-mediating nuclear translocation of VPAC1 produces a novel anti-apoptotic signal pathway, which may help to promote new drug development strategy targeting VPAC1.

  1. Cytokinin signaling regulates pavement cell morphogenesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Hongjiang Li; Tongda Xu; Deshu Lin; Mingzhang Wen; Mingtang Xie; Jér(o)me Duclercq; Agnieszka Bielach

    2013-01-01

    The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue.Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways.To identify additional components or mechanisms,we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern.Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines,the ahk3cre1 cytokinin receptor mutant,and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation,whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon.Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern.Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

  2. Electrical stimulation of neural stem cells mediated by humanized carbon nanotube composite made with extracellular matrix protein.

    Science.gov (United States)

    Kam, Nadine Wong Shi; Jan, Edward; Kotov, Nicholas A

    2009-01-01

    One of the key challenges to engineering neural interfaces is to minimize their immune response toward implanted electrodes. One potential approach is to manufacture materials that bear greater structural resemblance to living tissues and by utilizing neural stem cells. The unique electrical and mechanical properties of carbon nanotubes make them excellent candidates for neural interfaces, but their adoption hinges on finding approaches for "humanizing" their composites. Here we demonstrated the fabrication of layer-by-layer assembled composites from single-walled carbon nanotubes (SWNTs) and laminin, which is an essential part of human extracellular matrix. Laminin-SWNT thin films were found to be conducive to neural stem cells (NSC) differentiation and suitable for their successful excitation. We observed extensive formation of functional neural network as indicated by the presence of synaptic connections. Calcium imaging of the NSCs revealed generation of action potentials upon the application of a lateral current through the SWNT substrate. These results indicate that the protein-SWNT composite can serve as materials foundation of neural electrodes with chemical structure better adapted with long-term integration with the neural tissue.

  3. Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media.

    Science.gov (United States)

    Short, Kirsty R; von Köckritz-Blickwede, Maren; Langereis, Jeroen D; Chew, Keng Yih; Job, Emma R; Armitage, Charles W; Hatcher, Brandon; Fujihashi, Kohtaro; Reading, Patrick C; Hermans, Peter W; Wijburg, Odilia L; Diavatopoulos, Dimitri A

    2014-01-01

    Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

  4. Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

    Directory of Open Access Journals (Sweden)

    Kristine Porter

    Full Text Available Cells in the trabecular meshwork (TM, a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment. Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB. Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  5. Striatal adenosine signaling regulates EAAT2 and astrocytic AQP4 expression and alcohol drinking in mice.

    Science.gov (United States)

    Lee, Moonnoh R; Ruby, Christina L; Hinton, David J; Choi, Sun; Adams, Chelsea A; Young Kang, Na; Choi, Doo-Sup

    2013-02-01

    Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.

  6. Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles.

    Science.gov (United States)

    Vituret, Cyrielle; Gallay, Kathy; Confort, Marie-Pierre; Ftaich, Najate; Matei, Constantin I; Archer, Fabienne; Ronfort, Corinne; Mornex, Jean-François; Chanson, Marc; Di Pietro, Attilio; Boulanger, Pierre; Hong, Saw See

    2016-02-01

    Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA(GFP-CFTR)) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR). Both microvesicles and exosomes had the capacity to package and deliver the GFP-CFTR glycoprotein and mRNA(GFP-CFTR) to target cells in a dose-dependent manner. Homologous versus heterologous EV-to-cell transfer was studied, and it appeared that the cellular uptake of EVs was significantly more efficient in homologous transfer. The incubation of CF15 cells, a nasal epithelial cell line homozygous for the ΔF508 CFTR mutation, with microvesicles or exosomes loaded with GFP-CFTR resulted in the correction of the CFTR function in CF cells in a dose-dependent manner. A time-course analysis of EV-transduced CF cells suggested that CFTR transferred as mature glycoprotein was responsible for the CFTR-associated channel activity detected at early times posttransduction, whereas GFP-CFTR translated from exogenous mRNA(GFP-CFTR) was responsible for the CFTR function at later times. Collectively, this study showed the potential application of microvesicles and exosomes as vectors for CFTR transfer and functional correction of the genetic defect in human CF cells.

  7. The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.

    Science.gov (United States)

    Gilbert, Hannah E; Asokan, Rengasamy; Holers, V Michael; Perkins, Stephen J

    2006-10-01

    Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.

  8. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    Energy Technology Data Exchange (ETDEWEB)

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. (Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill (United States))

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  9. NMDA-mediated and Self-induced Bdnf Exon IV Transcriptions are Differentially Regulated in Cultured Cortical Neurons

    OpenAIRE

    Zheng, Fei; Wang, Hongbing

    2009-01-01

    Activity-dependent transcriptional up-regulation of bdnf (brain-derived neurotrophic factor) is involved in regulating many aspects of neuronal functions. The NMDA (N-methyl-D-aspartic acid)-mediated and BDNF-mediated exon IV transcription may represent mechanistically different responses, and relevant to activity-dependent changes in neurons. We found that the activities of ERK (extracellular signal-regulated kinase), CaM KII/IV (calmodulin-dependent protein kinase II and IV), PI3K (phosphoi...

  10. Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ in diabetic nephropathy.

    Science.gov (United States)

    Duan, Li-Jun; Ding, Min; Hou, Li-Jun; Cui, Yuan-Tao; Li, Chun-Jun; Yu, De-Min

    2017-03-11

    Long noncoding RNA taurine-upregulated gene 1 (lncRNA TUG1) has been reported to play a key role in the progression of diabetic nephropathy (DN). However, the role of lncRNA TUG1 in the regulation of diabetic nephropathy remains largely unknown. The aim of the present study is to identify the regulation of lncRNA TUG1 on extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ, and investigate the underlying mechanisms in progression of DN. Microarray was performed to screen differentially expressed miRNAs in db/db DN mice. Afterwards, computational prediction programs (TargetScan, miRanda, PicTar and miRGen) was applied to predict the target gene of miRNAs. The complementary binding of miRNA and lncRNA was assessed by luciferase assays. Protein and mRNA expression were detected by western blot and real time quantitate PCR. MiRNA-377 was screened by miRNA microarray and differentially up-regulated in db/db DN mice. PPARγ was predicted to be the target of miR-377 and the prediction was verified by luciferase assays. Expression of miR-377 was up-regulated in mesangial cell treated with high glucose (25 mM), and overexpression of miR-377 inhibited PPARγ expression and promoted PAI-1 and TGF-β1 expression. The expression of TUG1 antagonized the effect of miR-377 on the downregulation of its target PPARγ and inhibited extracellular matrix accumulation, including PAI-1, TGF-β1, fibronectin (FN) and collagen IV (Col IV), induced by high glucose. LncRNA TUG1 acts as an endogenous sponge of miR-377 and downregulates miR-377 expression levels, and thereby relieving the inhibition of its target gene PPARγ and alleviates extracellular matrix accumulation of mesangial cells, which provides a novel insight of diabetic nephropathy pathogenesis.

  11. Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

    Science.gov (United States)

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon D; Kapur, Reuben; Ingram, David A

    2014-01-01

    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.

  12. G protein-coupled receptor 120 signaling regulates ghrelin secretion in vivo and in vitro.

    Science.gov (United States)

    Gong, Zhi; Yoshimura, Makoto; Aizawa, Sayaka; Kurotani, Reiko; Zigman, Jeffrey M; Sakai, Takafumi; Sakata, Ichiro

    2014-01-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by ∼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.

  13. AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

    Science.gov (United States)

    Jung, Chiau-Jing; Hsu, Ron-Bin; Shun, Chia-Tung; Hsu, Chih-Chieh; Chia, Jean-San

    2017-09-01

    Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis. Copyright © 2017 American Society for Microbiology.

  14. Paradoxical signaling regulates structural plasticity in dendritic spines

    OpenAIRE

    2016-01-01

    Transient spine enlargement (3- to 5-min timescale) is an important event associated with the structural plasticity of dendritic spines. Many of the molecular mechanisms associated with transient spine enlargement have been identified experimentally. Here, we use a systems biology approach to construct a mathematical model of biochemical signaling and actin-mediated transient spine expansion in response to calcium influx caused by NMDA receptor activation. We have identified that a key featur...

  15. Cannabinoid receptor signaling regulates liver development and metabolism.

    Science.gov (United States)

    Liu, Leah Y; Alexa, Kristen; Cortes, Mauricio; Schatzman-Bone, Stephanie; Kim, Andrew J; Mukhopadhyay, Bani; Cinar, Resat; Kunos, George; North, Trista E; Goessling, Wolfram

    2016-02-15

    Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

  16. Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

    Science.gov (United States)

    Mekhdjian, Armen H; Kai, FuiBoon; Rubashkin, Matthew G; Prahl, Louis S; Przybyla, Laralynne M; McGregor, Alexandra L; Bell, Emily S; Barnes, J Matthew; DuFort, Christopher C; Ou, Guanqing; Chang, Alice C; Cassereau, Luke; Tan, Steven J; Pickup, Michael W; Lakins, Jonathan N; Ye, Xin; Davidson, Michael W; Lammerding, Jan; Odde, David J; Dunn, Alexander R; Weaver, Valerie M

    2017-06-01

    Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome. © 2017 Mekhdjian, Kai, Rubashkin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. Canonical Wnt Signaling Regulates Atrioventricular Junction Programming and Electrophysiological Properties

    Science.gov (United States)

    Gillers, Benjamin S; Chiplunkar, Aditi; Aly, Haytham; Valenta, Tomas; Basler, Konrad; Christoffels, Vincent M.; Efimov, Igor R; Boukens, Bastiaan J; Rentschler, Stacey

    2014-01-01

    Rationale Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. Objective To determine the role of canonical Wnt signaling in the myocardium during AVC development. Methods and Results We utilized a novel allele of β-catenin that preserves β-catenin’s cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiologic criteria. Aberrant AVC development can lead to ventricular preexcitation, a characteristic feature of Wolff-Parkinson-White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of β-catenin protein levels can rescue Notch-mediated ventricular preexcitation and dysregulated ion channel gene expression. Conclusions Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electrical programming upstream of Tbx3. Our data further suggests that ventricular preexcitation may require both morphologic patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue. PMID:25599332

  18. Nutritional status-dependent endocannabinoid signalling regulates the integration of rat visceral information.

    Science.gov (United States)

    Khlaifia, Abdessattar; Matias, Isabelle; Cota, Daniela; Tell, Fabien

    2017-06-01

    the extracellular signal-regulated kinase pathway. These data suggest a tight link between eCB-LTD in the NTS and nutritional status and shed light on the key role of eCB in the integration of visceral information. © 2017 Aix Marseille Université. The Journal of Physiology © 2017 The Physiological Society.

  19. Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    LI Jing; ZHANG Chuan-shan; L(U) Guo-dong; WANG Jun-hua; WEN Hao; YAN Gen-qiang; WEI Xu-fa; LIN Ren-yong

    2011-01-01

    Background Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs ofthe host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage.Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK)mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.Methods DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.Results We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.Conclusions We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and

  20. Xanthohumol inhibits the extracellular signal regulated kinase (ERK) signalling pathway and suppresses cell growth of lung adenocarcinoma cells.

    Science.gov (United States)

    Sławińska-Brych, Adrianna; Zdzisińska, Barbara; Dmoszyńska-Graniczka, Magdalena; Jeleniewicz, Witold; Kurzepa, Jacek; Gagoś, Mariusz; Stepulak, Andrzej

    2016-05-16

    Aberrant activation of the Ras/MEK/ERK signaling pathway has been frequently observed in non-small-cell lung carcinoma (NSCLC) and its important role in cancer progression and malignant transformation has been documented. Hence, the ERK1/2 kinase cascade becomes a potential molecular target in cancer treatment. Xanthohumol (XN, a prenylated chalcone derived from hope cones) is known to possess a broad spectrum of chemopreventive and anticancer activities. In our studies, the MTT and BrdU assays revealed that XN demonstrated greater antiproliferative activity against A549 lung adenocarcinoma cells than against the lung adenocarcinoma H1563 cell line. We observed that XN was able to suppress the activities of ERK1/2 and p90RSK kinases, followed by inhibition of phosphorylation and activation of the CREB protein. Additionally, the XN treatment of the cancer cells caused upregulation of key cell cycle regulators p53 and p21 as well as downregulation of cyclin D1. As a result, the cytotoxic effect of XN was attributed to the cell cycle arrest at G1 phase and induction of apoptosis indicated by increased caspase-3 activity. Thus, XN might be a promising anticancer drug candidate against lung carcinomas.

  1. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig;

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  2. Preconditioning with Azadirachta indica ameliorates cardiorenal dysfunction through reduction in oxidative stress and extracellular signal regulated protein kinase signalling

    Directory of Open Access Journals (Sweden)

    Temidayo Olutayo Omóbòwálé

    2016-10-01

    Conclusions: Together, A. indica and vitamin C prevented IRI-induced cardiorenal dysfunction via reduction in oxidative stress, improvement in antioxidant defence system and increase in the ERK1/2 expressions. Therefore, A. indica can be a useful chemopreventive agent in the prevention and treatment of conditions associated with intestinal ischaemia-reperfusion injury.

  3. Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

    Science.gov (United States)

    Rinne, Petteri; Rami, Martina; Nuutinen, Salla; Santovito, Donato; van der Vorst, Emiel P C; Guillamat-Prats, Raquel; Lyytikäinen, Leo-Pekka; Raitoharju, Emma; Oksala, Niku; Ring, Larisa; Cai, Minying; Hruby, Victor J; Lehtimäki, Terho; Weber, Christian; Steffens, Sabine

    2017-07-04

    The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse

  4. Lipid rafts as major platforms for signaling regulation in cancer.

    Science.gov (United States)

    Mollinedo, Faustino; Gajate, Consuelo

    2015-01-01

    Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient in vitro and in vivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as

  5. Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway.

    Science.gov (United States)

    Chakraborty, Sayan; Njah, Kizito; Pobbati, Ajaybabu V; Lim, Ying Bena; Raju, Anandhkumar; Lakshmanan, Manikandan; Tergaonkar, Vinay; Lim, Chwee Teck; Hong, Wanjin

    2017-03-07

    The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

  6. Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

    Directory of Open Access Journals (Sweden)

    Sayan Chakraborty

    2017-03-01

    Full Text Available The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

  7. Beta-adrenergic signals regulate cardiac differentiation of mouse embryonic stem cells via mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Yan, Lihui; Jia, Zhuqing; Cui, Jingjing; Yang, Hongtao; Yang, Huangtian; Zhang, Yongzhen; Zhou, Chunyan

    2011-08-01

    As embryonic stem cell-derived cardiomyocytes (ESC-CMs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of adrenergic and muscarinic receptors in mouse embryonic stem cells (ESCs). However, little is known about the role of these receptors in cardiac differentiation and development, which is critically important in cardiac physiology and pharmacology. Here, we demonstrated that a β-adrenergic receptor (β-AR) agonist significantly enhanced cardiac differentiation as indicated by a higher percentage of beating embryoid bodies and a higher expression level of cardiac markers. Application of β1-AR and β2-AR antagonists partly abolished the effect of the β-AR agonist. In addition, by administering selective inhibitors we found that the effect of β-AR was driven via p38 mitogen-activated protein kinase and extracellular-signal regulated kinase pathway. These findings suggest that ESCs are also a target for β-adrenergic regulation and β-adrenergic signaling plays a role in ESC cardiac differentiation.

  8. Extracellular calcium sensing and extracellular calcium signaling

    Science.gov (United States)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  9. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  10. Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum)

    OpenAIRE

    Phan, Anne Q.; Lee, Jangwoo; Oei, Michelle; Flath, Craig; Hwe, Caitlyn; Mariano, Rachele; Vu, Tiffany; Shu,Cynthia; Dinh, Andrew; Simkin, Jennifer; Muneoka, Ken; Bryant, Susan V.; Gardiner, David M

    2015-01-01

    Abstract Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain‐of‐function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneratio...

  11. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus.

    Science.gov (United States)

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N

    2015-03-01

    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus.

  12. Activation of P-glycoprotein (Pgp)-mediated drug efflux by extracellular acidosis: in vivo imaging with {sup 68}Ga-labelled PET tracer

    Energy Technology Data Exchange (ETDEWEB)

    Thews, Oliver; Dillenburg, Wolfgang [University Medicine Mainz, Institute of Physiology and Pathophysiology, Mainz (Germany); Fellner, Marco; Roesch, Frank [University of Mainz, Institute of Nuclear Chemistry, Mainz (Germany); Buchholz, Hans-Georg; Bausbacher, Nicole; Schreckenberger, Mathias [University Medicine Mainz, Department of Nuclear Medicine, Mainz (Germany)

    2010-10-15

    In vitro it has been shown that the functional activity of P-glycoprotein (Pgp), an important drug transporter responsible for multidrug resistance, can be strongly increased by extracellular acidosis. Here mitogen-activated protein kinases (MAPK) (p38, ERK1/2) seem to play an important role for signal transduction. However, it is unclear whether these effects are also relevant in vivo. With the newly developed PET tracer Schiff base-based {sup 68}Ga-MFL6.MZ the functional Pgp activity was visualized under acidic conditions and during inhibition of MAPKs non-invasively by means of microPET in rat tumours. Tumours were acidified either by inspiratory hypoxia (8% O{sub 2}) or by injection of lactic acid. Inhibitors of the MAPK were injected intratumourally. With increasing tumour volume the tumour pH changed from 7.0 to 6.7 and simultaneously the Pgp activity increased almost linearly. When the tumour was acidified by direct lactic acid injection the PET tracer uptake was reduced by 20% indicating a higher transport rate out of the cells. Changing the inspiratory O{sub 2} fraction to 8% dynamically led to a reduction of extracellular pH and in parallel to a decrease of tracer concentration. While inhibition of the p38 pathway reduced the Pgp transport rate, inhibition of ERK1/2 had practically no impact. An acidic extracellular environment significantly stimulates the Pgp activity. The p38 MAPK pathway plays an important role for Pgp regulation in vivo, whereas ERK1/2 is of minor importance. From these results new strategies for overcoming multidrug resistance (e.g. reducing tumour acidosis, inhibition of p38) may be developed. (orig.)

  13. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  14. Extracellular Matrix Proteins

    Directory of Open Access Journals (Sweden)

    Linda Christian Carrijo-Carvalho

    2012-01-01

    Full Text Available Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair.

  15. Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy.

    Science.gov (United States)

    Li, Jingjing; Chen, Kan; Li, Sainan; Feng, Jiao; Liu, Tong; Wang, Fan; Zhang, Rong; Xu, Shizan; Zhou, Yuqing; Zhou, Shunfeng; Xia, Yujing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2016-01-01

    Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis.

  16. Recognition of Aspergillus fumigatus hyphae by human plasmacytoid dendritic cells is mediated by dectin-2 and results in formation of extracellular traps.

    Directory of Open Access Journals (Sweden)

    Flávio V Loures

    2015-02-01

    Full Text Available Plasmacytoid dendritic cells (pDCs were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs. The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation.

  17. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.

  18. Black Soybean Seed Coat Extract Prevents Hydrogen Peroxide-Mediated Cell Death via Extracellular Signal-Related Kinase Signalling in HepG2 Cells.

    Science.gov (United States)

    Hashimoto, Naoto; Oki, Tomoyuki; Sasaki, Kazunori; Suda, Ikuo; Okuno, Shigenori

    2015-01-01

    Oxidative stress reduces cell viability and contributes to disease processes. Flavonoids including anthocyanins and proanthocyanidins reportedly induce intracellular antioxidant defence systems. Thus, in this study, we examined the antioxidant effects of a commercial extract from black soybean seed coats (BE), which are rich in anthocyanin and proanthocyanidin, and investigated the associated intracellular mechanisms in HepG2 cells. HepG2 cells treated with hydrogen peroxide (HPO) showed 60% viability, whereas pretreatment with BE-containing media for 2 h ameliorated HPO-mediated cell death by up to 90%. Pretreatment with BE for 2 h partially blocked HPO-mediated activation of ERK in HepG2 cells, and that for 1 h led to a 20% increase in intracellular total protein phosphatase (PP) activity, which is known to deactivate protein kinases. These results indicate that BE prevents HPO-mediated cell damage by inhibiting ERK signalling, potentially via PPs.

  19. Fungus-mediated preferential bioleaching of waste material such as fly - ash as a means of producing extracellular, protein capped, fluorescent and water soluble silica nanoparticles.

    Directory of Open Access Journals (Sweden)

    Shadab Ali Khan

    Full Text Available In this paper, we for the first time show the ability of the mesophilic fungus Fusarium oxysporum in the bioleaching of waste material such as Fly-ash for the extracellular production of highly crystalline and highly stable, protein capped, fluorescent and water soluble silica nanoparticles at ambient conditions. When the fungus Fusarium oxysporum is exposed to Fly-ash, it is capable of selectively leaching out silica nanoparticles of quasi-spherical morphology within 24 h of reaction. These silica nanoparticles have been completely characterized by UV-vis spectroscopy, Photoluminescence (PL, Transmission electron microscopy (TEM, X-ray diffraction (XRD, Fourier transform infrared spectroscopy (FTIR and Energy dispersive analysis of X-rays (EDAX.

  20. The PI3K/Akt Signaling Pathway Mediates the High Glucose-Induced Expression of Extracellular Matrix Molecules in Human Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Dong Qin

    2015-01-01

    Full Text Available Prolonged hyperglycemia is an important risk factor of the pathogenesis of diabetic retinopathy (DR. Extracellular matrix molecules, such as fibronectin, collagen IV, and laminin, are associated with fibrotic membranes. In this study, we investigated the expression of fibronectin, collagen IV, and laminin in RPE cells under high glucose conditions. Furthermore, we also detected the phosphorylation of protein kinase B (Akt under high glucose conditions in RPE cells. Our results showed that high glucose upregulated fibronectin, collagen IV, and laminin expression, and activated Akt in RPE cells. We also found that pretreatment with LY294002 (an inhibitor of phosphatidylinositol 3-kinase abolished high glucose-induced expression of fibronectin, collagen IV, and laminin in RPE cells. Thus, high glucose induced the expression of fibronectin, collagen IV, and laminin through PI3K/Akt signaling pathway in RPE cells, and the PI3K/Akt signaling pathway may contribute to the formation of fibrotic membrane during the development of DR.

  1. Inositol and Phosphatidylinositol Mediated Glucose Derepression, Gene Expression and Invertase Secretion in Yeasts

    Institute of Scientific and Technical Information of China (English)

    Zhen-Ming CHI; Jun-Feng LI; Xiang-Hong WANG; Shu-Min YAO

    2004-01-01

    Glucose repression occurs in many yeast species and some filamentous fungi, and it represses the expression and secretion of many intracellular and extracellular proteins. In recent years, it has been found that many biochemical reactions in yeast cells are mediated by phosphatidylinositol (PI)-type signaling pathway. However, little is known about the relationships between PI-type signaling and glucose repression,gene expression and invertase secretion in yeasts. Many evidences in our previous studies showed that glucose repression, invertase secretion, gene expression and cell growth were mediated by inositol and PI in Saccharomyces and Schizosaccharomyces. The elucidation of the new regulatory mechanisms of protein secretion, gene expression and glucose repression would be an entirely new aspect of inositol and PI-type signaling regulation in yeasts.

  2. Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy

    Directory of Open Access Journals (Sweden)

    Li J

    2016-02-01

    Full Text Available Jingjing Li,1 Kan Chen,1 Sainan Li,1 Jiao Feng,1 Tong Liu,1 Fan Wang,1 Rong Zhang,1,2 Shizan Xu,1,2 Yuqing Zhou,1,3 Shunfeng Zhou,1,3 Yujing Xia,1 Jie Lu,1 Yingqun Zhou,1 Chuanyong Guo1 1Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 2The First Clinical Medical College of Nanjing Medical University, Nanjing, 3Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, People’s Republic of China Abstract: Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis. Keywords: liver cirrhosis, hepatic stellate cells, bile duct ligation

  3. Extracellular matrix molecules and synaptic plasticity: immunomapping of intracellular and secreted Reelin in the adult rat brain.

    Science.gov (United States)

    Ramos-Moreno, Tania; Galazo, Maria J; Porrero, Cesar; Martínez-Cerdeño, Verónica; Clascá, Francisco

    2006-01-01

    Reelin, a large extracellular matrix glycoprotein, is secreted by several neuron populations in the developing and adult rodent brain. Secreted Reelin triggers a complex signaling pathway by binding lipoprotein and integrin membrane receptors in target cells. Reelin signaling regulates migration and dendritic growth in developing neurons, while it can modulate synaptic plasticity in adult neurons. To identify which adult neural circuits can be modulated by Reelin-mediated signaling, we systematically mapped the distribution of Reelin in adult rat brain using sensitive immunolabeling techniques. Results show that the distribution of intracellular and secreted Reelin is both very widespread and specific. Some interneuron and projection neuron populations in the cerebral cortex contain Reelin. Numerous striatal neurons are weakly immunoreactive for Reelin and these cells are preferentially located in striosomes. Some thalamic nuclei contain Reelin-immunoreactive cells. Double-immunolabeling for GABA and Reelin reveals that the Reelin-immunoreactive cells in the visual thalamus are the intrinsic thalamic interneurons. High local concentrations of extracellular Reelin selectively outline several dendrite spine-rich neuropils. Together with previous mRNA data, our observations suggest abundant axoplasmic transport and secretion in pathways such as the retino-collicular tract, the entorhino-hippocampal ('perforant') path, the lateral olfactory tract or the parallel fiber system of the cerebellum. A preferential secretion of Reelin in these neuropils is consistent with reports of rapid, activity-induced structural changes in adult brain circuits.

  4. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    Directory of Open Access Journals (Sweden)

    Xiaofei Cheng

    2016-01-01

    Full Text Available Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM. Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9, matrix metalloproteinase 3 (MMP-3, and tissue inhibitor of metalloproteinase 1 (TIMP-1, was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  5. Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Phan, Anne Q; Lee, Jangwoo; Oei, Michelle; Flath, Craig; Hwe, Caitlyn; Mariano, Rachele; Vu, Tiffany; Shu, Cynthia; Dinh, Andrew; Simkin, Jennifer; Muneoka, Ken; Bryant, Susan V; Gardiner, David M

    2015-08-01

    Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain-of-function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position-specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position-specific, developmental-stage-specific, and heparan sulfate-dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals.

  6. Interleukin-1-mediated effects of normal oral keratinocytes and head and neck squamous carcinoma cells on extracellular matrix related gene expression in fibroblasts.

    Science.gov (United States)

    Hakelius, Malin; Koskela, Anita; Reyhani, Vahid; Ivarsson, Mikael; Grenman, Reidar; Rubin, Kristofer; Gerdin, Bengt; Nowinski, Daniel

    2012-12-01

    The composition of tumor stroma and the activity of tumor associated fibroblasts are important for tumor growth. Interactions between carcinoma cells and fibroblasts regulate the turnover of extracellular matrix (ECM). Here, the in vitro effects of oral squamous cell carcinoma (SCC) cells (UT-SCC-30 and UT-SCC-87) on fibroblast expression of genes for ECM components and connective tissue growth factor (CTGF/CCN2), were compared to those of normal oral keratinocytes (NOK). Cocultures with fibroblasts in collagen gels and keratinocytes with the two cell types separated by a semi permeable membrane were used, and relative gene expression was measured with real-time PCR. All investigated genes were regulated by NOK and the SCCs. The downregulation of pro-collagens α1(I) and α1(III) was more pronounced in cocultures with NOK, while the expression of CCN2 and fibronectin was downregulated by both NOK and the SCCs to a similar extent. UT-SCC-87, but not UT-SCC-30, secreted significantly more IL-1α than NOK. A recombinant interleukin-1 receptor antagonist reversed many of the observed effects on fibroblast gene expression suggesting involvement of IL-1 in cocultures with NOK as well as with SCCs. The observed differential effects on fibroblast gene expression suggest that NOK are more antifibrotic compared to UT-SCC-30 and UT-SCC-87. These findings may contribute to a better understanding of the mechanisms behind ECM turnover in tumors. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis.

    Science.gov (United States)

    Alvarez-Llamas, Gloria; Martín-Rojas, Tatiana; de la Cuesta, Fernando; Calvo, Enrique; Gil-Dones, Felix; Dardé, Veronica M; Lopez-Almodovar, Luis F; Padial, Luis R; Lopez, Juan-Antonio; Vivanco, Fernando; Barderas, Maria G

    2013-09-01

    One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.

  8. Cardiac-Restricted IGF-1Ea Overexpression Reduces the Early Accumulation of Inflammatory Myeloid Cells and Mediates Expression of Extracellular Matrix Remodelling Genes after Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Enrique Gallego-Colon

    2015-01-01

    Full Text Available Strategies to limit damage and improve repair after myocardial infarct remain a major therapeutic goal in cardiology. Our previous studies have shown that constitutive expression of a locally acting insulin-like growth factor-1 Ea (IGF-1Ea propeptide promotes functional restoration after cardiac injury associated with decreased scar formation. In the current study, we investigated the underlying molecular and cellular mechanisms behind the enhanced functional recovery. We observed improved cardiac function in mice overexpressing cardiac-specific IGF-1Ea as early as day 7 after myocardial infarction. Analysis of gene transcription revealed that supplemental IGF-1Ea regulated expression of key metalloproteinases (MMP-2 and MMP-9, their inhibitors (TIMP-1 and TIMP-2, and collagen types (Col 1α1 and Col 1α3 in the first week after injury. Infiltration of inflammatory cells, which direct the remodelling process, was also altered; in particular there was a notable reduction in inflammatory Ly6C+ monocytes at day 3 and an increase in anti-inflammatory CD206+ macrophages at day 7. Taken together, these results indicate that the IGF-1Ea transgene shifts the balance of innate immune cell populations early after infarction, favouring a reduction in inflammatory myeloid cells. This correlates with reduced extracellular matrix remodelling and changes in collagen composition that may confer enhanced scar elasticity and improved cardiac function.

  9. Glutamate transporter activity promotes enhanced Na+/K+-ATPase -mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian R; Holm, Rikke; Vilsen, Bente;

    2016-01-01

    , in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether...... astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+ /K+ -ATPase was not fully saturated at basal astrocytic [Na+ ]i , irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na...

  10. Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

    Science.gov (United States)

    Bordeleau, François; Myrand Lapierre, Marie-Eve; Sheng, Yunlong; Marceau, Normand

    2012-01-01

    Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells. PMID:22685604

  11. Keratin 8/18 regulation of cell stiffness-extracellular matrix interplay through modulation of Rho-mediated actin cytoskeleton dynamics.

    Directory of Open Access Journals (Sweden)

    François Bordeleau

    Full Text Available Cell mechanical activity generated from the interplay between the extracellular matrix (ECM and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18, hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells.

  12. Therapeutic Development of Mesenchymal Stem Cells or Their Extracellular Vesicles to Inhibit Autoimmune-Mediated Inflammatory Processes in Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Juhi Sharma

    2017-05-01

    Full Text Available Since being discovered over half a century ago, mesenchymal stem cells (MSCs have been investigated extensively to characterize their cellular and physiological influences. MSCs have been shown to possess immunosuppressive capacity through inhibiting lymphocyte activation/proliferation and proinflammatory cytokine secretion while simultaneously demonstrating limited allogenic reactivity, which subsequently led to the evaluation of therapeutic feasibility to treat inflammatory diseases. Although regulatory constraints have restricted MSC development pharmacologically, limited clinical studies have shown encouraging results using MSC infusions to treat systemic lupus erythematosus (SLE; but, more trials will have to be performed to conclusively determine the clinical efficacy of MSCs to treat SLE. Moreover, there are some data to suggest that MSCs possess tumorigenic potential and that the immunosuppressive influence can be dramatically affected by both donor variability and ex vivo expansion. Given that recent studies have found that the immunosuppressive effects of MSCs are a result, at least in part, to extracellular vesicle (EV secretion, the use of MSC-derived EVs has been suggested as a cell-free therapeutic alternative. Despite the positive data observed using EVs isolated from human MSCs to suppress inflammatory responses in vitro and in inhibiting autoimmune disease pathogenesis in preclinical work, there are no studies to date examining EVs from MSCs to treat SLE in humans or animal models. Considering that EVs are not subject to the strict regulatory constraints of stem cell-based pharmacological development and are more readily standardized with regard to industrial-scale production and storage, this review outlines the anti-inflammatory biology of MSCs and the scientific evidence supporting the potential use of EVs derived from human MSCs to treat patients with SLE.

  13. The syndecan-4/protein kinase Cα pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo.

    Science.gov (United States)

    Corti, Federico; Finetti, Federica; Ziche, Marina; Simons, Michael

    2013-05-03

    Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4(-/-) mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4(-/-) endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα(-/-) mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.

  14. Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response

    Directory of Open Access Journals (Sweden)

    V. A. Sergeeva

    2017-01-01

    Full Text Available We have hypothesized that the adaptive response to low doses of ionizing radiation (IR is mediated by oxidized cell-free DNA (cfDNA fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production, induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs with low doses of IR (10 cGy leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.

  15. Differences in signal activation by LH and hCG are mediated by the LH/CG receptor`s extracellular hinge region

    Directory of Open Access Journals (Sweden)

    Paul eGrzesik

    2015-09-01

    Full Text Available The human lutropin/choriogonadotropin receptor (LHCGR can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG - secreted by the placenta, and lutropin (LH - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich repeat domain (LRRD, as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting mutations. These helix preserving modifications showed no effect on hormone induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10 deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region s. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region

  16. C/EBP β Mediates Endoplasmic Reticulum Stress Regulated Inflammatory Response and Extracellular Matrix Degradation in LPS-Stimulated Human Periodontal Ligament Cells

    Directory of Open Access Journals (Sweden)

    Yudi Bai

    2016-03-01

    Full Text Available Periodontitis is an oral inflammatory disease that not only affects the integrity of local tooth-supporting tissues but also impacts systemic health. A compositional shift in oral microbiota has been considered as the main cause of periodontitis; however, the potential mechanism has not been fully defined. Herein, we investigated the role of CCAAT/enhancer-binding protein β (C/EBP β, a member of the C/EBP family of transcription factors, in human periodontal ligament cells (hPDLCs exposed to Porphyromonas gingivalis (P. gingivalis lipopolysaccharide (LPS. RT-PCR and Western blotting analysis showed that the expression of C/EBP β was significantly increased in hPDLCs stimulated with LPS stimuli. Overexpression of C/EBP β by the recombinant adenoviral vector pAd/C/EBP β markedly increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and matrix metalloproteinases (MMP-8 and -9 in hPDLCs in response to LPS. Furthermore, the activation of endoplasmic reticulum (ER stress was confirmed in LPS-stimulated hPDLCs by measuring the expression of the ER stress marker molecules protein kinase-like ER kinase (PERK, eIF2α, GRP78/Bip, and C/EBP homologous protein (CHOP. The ER stress inhibitor salubrinal repressed, but inducer tunicamycin enhanced, the production of IL-6, IL-8, MMP-8, and MMP-9 in hPDLCs. Additionally, ER stress inducer tunicamycin significantly increased the expression level of C/EBP β in hPDLCs. Blocking of C/EBP β by siRNA resulted in a significant decrease in the secretion of IL-6 and IL-8 and expression of MMP-8 and MMP-9 induced by tunicamycin treatment in hPDLCs. Taken together, ER stress appears to play a regulatory role in the inflammatory response and extracellular matrix (ECM degradation in hPDLCs in response to LPS stimuli by activating C/EBP β expression. This enhances our understanding of human periodontitis pathology.

  17. C/EBP β Mediates Endoplasmic Reticulum Stress Regulated Inflammatory Response and Extracellular Matrix Degradation in LPS-Stimulated Human Periodontal Ligament Cells.

    Science.gov (United States)

    Bai, Yudi; Wei, Yi; Wu, Lian; Wei, Jianhua; Wang, Xiaojing; Bai, Yuxiang

    2016-03-22

    Periodontitis is an oral inflammatory disease that not only affects the integrity of local tooth-supporting tissues but also impacts systemic health. A compositional shift in oral microbiota has been considered as the main cause of periodontitis; however, the potential mechanism has not been fully defined. Herein, we investigated the role of CCAAT/enhancer-binding protein β (C/EBP β), a member of the C/EBP family of transcription factors, in human periodontal ligament cells (hPDLCs) exposed to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). RT-PCR and Western blotting analysis showed that the expression of C/EBP β was significantly increased in hPDLCs stimulated with LPS stimuli. Overexpression of C/EBP β by the recombinant adenoviral vector pAd/C/EBP β markedly increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and matrix metalloproteinases (MMP)-8 and -9 in hPDLCs in response to LPS. Furthermore, the activation of endoplasmic reticulum (ER) stress was confirmed in LPS-stimulated hPDLCs by measuring the expression of the ER stress marker molecules protein kinase-like ER kinase (PERK), eIF2α, GRP78/Bip, and C/EBP homologous protein (CHOP). The ER stress inhibitor salubrinal repressed, but inducer tunicamycin enhanced, the production of IL-6, IL-8, MMP-8, and MMP-9 in hPDLCs. Additionally, ER stress inducer tunicamycin significantly increased the expression level of C/EBP β in hPDLCs. Blocking of C/EBP β by siRNA resulted in a significant decrease in the secretion of IL-6 and IL-8 and expression of MMP-8 and MMP-9 induced by tunicamycin treatment in hPDLCs. Taken together, ER stress appears to play a regulatory role in the inflammatory response and extracellular matrix (ECM) degradation in hPDLCs in response to LPS stimuli by activating C/EBP β expression. This enhances our understanding of human periodontitis pathology.

  18. Extracellular regulated kinase phosphorylates mitofusin 1 to control mitochondrial morphology and apoptosis.

    Science.gov (United States)

    Pyakurel, Aswin; Savoia, Claudia; Hess, Daniel; Scorrano, Luca

    2015-04-16

    Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1(T562). Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis.

  19. Extracellular Regulated Kinase Phosphorylates Mitofusin 1 to Control Mitochondrial Morphology and Apoptosis

    Science.gov (United States)

    Pyakurel, Aswin; Savoia, Claudia; Hess, Daniel; Scorrano, Luca

    2015-01-01

    Summary Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1T562. Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis. PMID:25801171

  20. Involvement of extracellular matrix constituents in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  1. Dopamine Signaling Regulates Fat Content through β-Oxidation in Caenorhabditis elegans

    Science.gov (United States)

    Barros, Alexandre Guimarães de Almeida; Bridi, Jessika Cristina; de Souza, Bruno Rezende; de Castro Júnior, Célio; de Lima Torres, Karen Cecília; Malard, Leandro; Jorio, Ado; de Miranda, Débora Marques; Ashrafi, Kaveh; Romano-Silva, Marco Aurélio

    2014-01-01

    The regulation of energy balance involves an intricate interplay between neural mechanisms that respond to internal and external cues of energy demand and food availability. Compelling data have implicated the neurotransmitter dopamine as an important part of body weight regulation. However, the precise mechanisms through which dopamine regulates energy homeostasis remain poorly understood. Here, we investigate mechanisms through which dopamine modulates energy storage. We showed that dopamine signaling regulates fat reservoirs in Caenorhabditis elegans. We found that the fat reducing effects of dopamine were dependent on dopaminergic receptors and a set of fat oxidation enzymes. Our findings reveal an ancient role for dopaminergic regulation of fat and suggest that dopamine signaling elicits this outcome through cascades that ultimately mobilize peripheral fat depots. PMID:24465759

  2. Wnt signaling regulates multipolar-to-bipolar transition of migrating neurons in the cerebral cortex.

    Science.gov (United States)

    Boitard, Michael; Bocchi, Riccardo; Egervari, Kristof; Petrenko, Volodymyr; Viale, Beatrice; Gremaud, Stéphane; Zgraggen, Eloisa; Salmon, Patrick; Kiss, Jozsef Z

    2015-03-03

    The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration. In addition, we show that reduced canonical Wnt signaling is triggered cell autonomously by time-dependent expression of Wnt5A and activation of non-canonical signaling. We identify ephrin-B1 as a canonical Wnt-signaling-regulated target in control of the multipolar-to-bipolar switch. These findings highlight the critical role of Wnt signaling activity in neuronal positioning during cortical development.

  3. Is cholesterol and amyloid-β stress induced CD147 expression a protective response? Evidence that extracellular cyclophilin a mediated neuroprotection is reliant on CD147.

    Science.gov (United States)

    Kanyenda, Limbikani J; Verdile, Guiseppe; Martins, Ralph; Meloni, Bruno P; Chieng, Joanne; Mastaglia, Francis; Laws, Simon M; Anderton, Ryan S; Boulos, Sherif

    2014-01-01

    The CD147 protein is a ubiquitous multifunctional membrane receptor. Expression of CD147, which is regulated by sterol carrier protein, reportedly modulates amyloid-β (Aβ), the neurotoxic peptide implicated in neuronal degeneration in Alzheimer's disease (AD). Given that high fat/cholesterol is linked to amyloid deposition in AD, we investigated if cholesterol and/or Aβ can alter CD147 expression in rat cortical neuronal cultures. Water-soluble cholesterol and Aβ42 dose-dependently increased CD147 protein expression, but reduced FL-AβPP protein expression. Cholesterol and Aβ42 treatment also increased lactate dehydrogenase release but to varying degrees. Upregulation of CD147 expression was probably mediated by oxidative stress, as H2O2 (3 μM) also induced CD147 protein expression in neuronal cultures. In light of these findings, we investigated if CD147 induction was cytoprotective, a compensatory response to injury, or alternatively, a cell death signal. To this end, we used recombinant adenovirus to overexpress human CD147 (in SH-SY5Y cells and primary cortical neurons), and pre-treated cultures with or without recombinant cyclophilin A (rCYPA) protein, prior to Aβ42 exposure. We showed that increased CD147 expression protected against Aβ42, only when rCYPA protein was added to neuronal cultures. Together, our findings reveal potentially important relationships between cholesterol loading, CD147 expression, Aβ toxicity, and the putative involvement of CYPA protein in neuroprotection in AD.

  4. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    Science.gov (United States)

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

  5. Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

    NARCIS (Netherlands)

    Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A; Del Portillo, Hernando A; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Görgens, André; Gupta, Ramesh C; Hendrix, An; Hermann, Dirk M; Hill, Andrew F; Hochberg, Fred; Horn, Peter A; de Kleijn, Dominique|info:eu-repo/dai/nl/30481489X; Kordelas, Lambros; Kramer, Boris W; Krämer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J; Lim, Sai Kiang; Lötvall, Jan; Maguire, Casey A; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G; Rivera, Francisco J; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke|info:eu-repo/dai/nl/074327941; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W M|info:eu-repo/dai/nl/256594783; Wauben, Marca; Andaloussi, Samir El; Théry, Clotilde; Rohde, Eva; Giebel, Bernd

    2015-01-01

    Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information

  6. Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

    NARCIS (Netherlands)

    Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A; Del Portillo, Hernando A; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Görgens, André; Gupta, Ramesh C; Hendrix, An; Hermann, Dirk M; Hill, Andrew F; Hochberg, Fred; Horn, Peter A; de Kleijn, Dominique; Kordelas, Lambros; Kramer, Boris W; Krämer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J; Lim, Sai Kiang; Lötvall, Jan; Maguire, Casey A; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G; Rivera, Francisco J; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W M; Wauben, Marca; Andaloussi, Samir El; Théry, Clotilde; Rohde, Eva; Giebel, Bernd

    2015-01-01

    Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information fro

  7. Wnt Signaling Regulates Multipolar-to-Bipolar Transition of Migrating Neurons in the Cerebral Cortex

    Directory of Open Access Journals (Sweden)

    Michael Boitard

    2015-03-01

    Full Text Available The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration. In addition, we show that reduced canonical Wnt signaling is triggered cell autonomously by time-dependent expression of Wnt5A and activation of non-canonical signaling. We identify ephrin-B1 as a canonical Wnt-signaling-regulated target in control of the multipolar-to-bipolar switch. These findings highlight the critical role of Wnt signaling activity in neuronal positioning during cortical development.

  8. Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Tim N. Beck

    2016-07-01

    Full Text Available Anti-Müllerian hormone (AMH and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β/bone morphogenetic protein (BMP signaling and epithelial-mesenchymal transition (EMT in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3 also used by the type II receptor for BMP (BMPR2. AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC. AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90 inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.

  9. (Prorenin receptor triggers distinct angiotensin II-independent extracellular matrix remodeling and deterioration of cardiac function.

    Directory of Open Access Journals (Sweden)

    Anne-Mari Moilanen

    Full Text Available BACKGROUND: Activation of the renin-angiotensin-system (RAS plays a key pathophysiological role in heart failure in patients with hypertension and myocardial infarction. However, the function of (prorenin receptor ((PRR is not yet solved. We determined here the direct functional and structural effects of (PRR in the heart. METHODOLOGY/PRINCIPAL FINDINGS: (PRR was overexpressed by using adenovirus-mediated gene delivery in normal adult rat hearts up to 2 weeks. (PRR gene delivery into the anterior wall of the left ventricle decreased ejection fraction (P<0.01, fractional shortening (P<0.01, and intraventricular septum diastolic and systolic thickness, associated with approximately 2-fold increase in left ventricular (PRR protein levels at 2 weeks. To test whether the worsening of cardiac function and structure by (PRR gene overexpression was mediated by angiotensin II (Ang II, we infused an AT(1 receptor blocker losartan via osmotic minipumps. Remarkably, cardiac function deteriorated in losartan-treated (PRR overexpressing animals as well. Intramyocardial (PRR gene delivery also resulted in Ang II-independent activation of extracellular-signal-regulated kinase1/2 phosphorylation and myocardial fibrosis, and the expression of transforming growth factor-β1 and connective tissue growth factor genes. In contrast, activation of heat shock protein 27 phosphorylation and apoptotic cell death by (PRR gene delivery was Ang II-dependent. Finally, (PRR overexpression significantly increased direct protein-protein interaction between (PRR and promyelocytic zinc-finger protein. CONCLUSIONS/SIGNIFICANCE: These results indicate for the first time that (PRR triggers distinct Ang II-independent myocardial fibrosis and deterioration of cardiac function in normal adult heart and identify (PRR as a novel therapeutic target to optimize RAS blockade in failing hearts.

  10. Microwave-mediated extracellular synthesis of metallic silver and zinc oxide nanoparticles using macro-algae (Gracilaria edulis) extracts and its anticancer activity against human PC3 cell lines.

    Science.gov (United States)

    Priyadharshini, Ramaramesh Indra; Prasannaraj, Govindaraj; Geetha, Natesan; Venkatachalam, Perumal

    2014-12-01

    A rapid and novel microwave-mediated protocol was established for extracellular synthesis of metallic silver (Ag) and zinc oxide (ZnO) nanoparticles using the extracts of macro-algae Gracilaria edulis (GE) and also examined its anticancer activity against human prostate cancer cell lines (PC3). The formation of silver nanoparticles (GEAgNPs) and zinc oxide nanoparticles (GEZnONPs) in the reaction mixture was determined by ultraviolet-visible spectroscopy. The synthesized Ag and ZnO nanoparticles were characterized by X-ray diffraction, Fourier transform infra-red spectroscopy, energy dispersive X-ray, and field emission scanning electron microscopy. The silver and zinc oxide nanoparticles were spherical and rod-shaped, respectively. Cell viability assays were carried out to determine the cytotoxic effects of AgNPs and ZnONPs against PC3 and normal African monkey kidney (VERO) cell line. The inhibitory concentration values were found to be 39.60, 28.55, 53.99 μg/mL and 68.49, 88.05, 71.98 μg/mL against PC3 cells and Vero cells for AgNPs, ZnONPs, and aqueous G. edulis extracts, respectively, at 48 h incubation period. As evidenced by acridine orange/ethidium bromide staining, the percentage of the apoptotic bodies was found to be 62 and 70 % for AgNPs and ZnONPs, respectively. The present results strongly suggest that the synthesized ZnONPs showed an effective anticancer activity against PC3 cell lines than AgNPs.

  11. Endogenous nitric oxide induces activation of apoptosis signal-regulating kinase 1 via S-nitrosylation in rat hippocampus during cerebral ischemia-reperfusion.

    Science.gov (United States)

    Liu, D-H; Yuan, F-G; Hu, S-Q; Diao, F; Wu, Y-P; Zong, Y-Y; Song, T; Li, C; Zhang, G-Y

    2013-01-15

    Apoptosis signal-regulating kinase 1 (ASK1) is a general mediator of cell death in response to a variety of stimuli, including reactive oxygen species, tumor necrosis factor α, lipopolysaccharide, endoplasmic reticulum stress, calcium influx and ischemia. Here we reported ASK1 was activated by nitric oxide (NO) through S-nitrosylation during cerebral ischemia-reperfusion. The reagents that abrogate neuronal nitric oxide synthase (nNOS) activity such as nNOS inhibitor 7NI and N-methyl-D-aspartate receptor antagonist MK801 prevented ASK1 activation via decreasing ASK1 S-nitrosylation. In HEK293 cells, over-expressed ASK1 could be S-nitrosylated by both exogenous and endogenous NO and Cys869 was identified as the site of ASK1 S-nitrosylation. S-nitrosylation increased the level of ASK1 phosphorylation at Thr845, which represents ASK1 activation. Our results further confirmed that S-nitrosylation led to the increment of ASK1 dimerization. S-nitrosylation of ASK1 also activated the downstream JNK signaling and JNK-mediated nucleic pathway. The exogenous NO (SNP and GSNO) reversed the effect of endogenous NO by suppressing S-nitrosylation of ASK1 and exerted neuroprotection during ischemia-reperfusion. These results suggest that inhibiting ASK1 S-nitrosylation may be a novel approach for stroke therapy. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Notch-RBP-J signaling regulates the mobilization and function of endothelial progenitor cells by dynamic modulation of CXCR4 expression in mice.

    Directory of Open Access Journals (Sweden)

    Lin Wang

    Full Text Available Bone marrow (BM-derived endothelial progenitor cells (EPC have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.

  13. Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

    Science.gov (United States)

    Eaton, G J; Zhang, Q-S; Diallo, C; Matsuzawa, A; Ichijo, H; Steinbeck, M J; Freeman, T A

    2014-11-13

    Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has

  14. Inhibitor of Apoptosis Signal-Regulating Kinase 1 Protects Against Acetaminophen-induced Liver Injury

    Science.gov (United States)

    Xie, Yuchao; Ramachandran, Anup; Breckenridge, David G.; Liles, John T.; Lebofsky, Margitta; Farhood, Anwar; Jaeschke, Hartmut

    2015-01-01

    Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affected the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. PMID:25818599

  15. Differential expression of interleukin-1/Toll-like receptor signaling regulators in microscopic and ulcerative colitis.

    Science.gov (United States)

    Günaltay, Sezin; Nyhlin, Nils; Kumawat, Ashok Kumar; Tysk, Curt; Bohr, Johan; Hultgren, Olof; Hultgren Hörnquist, Elisabeth

    2014-09-14

    To investigate Toll-like receptor (TLR) signaling regulators in microscopic and ulcerative colitis patients. Total RNA and microRNA were isolated from fresh frozen colonic biopsies of non-inflamed controls and patients with active or in-remission collagenous colitis (CC), lymphocytic colitis (LC), or ulcerative colitis (UC). We compared expressions of interleukin-1 receptor-associated kinase (IRAK)-2, IRAK-M, interleukin (IL)-37, microRNA (miR)-146a, miR-155, and miR-21 using quantitative real time reverse transcription polymerase chain reaction. IRAK-M expression was increased in LC patients with active disease in histopathological remission (LC-HR; P = 0.02) and UC patients (P = 0.01), but no differences in IRAK-2 expression were detected compared to controls. miR-146a, -155 and -21 expressions were increased in LC-HR (P = 0.04, 0.07, and 0.004) and UC (P = 0.02, 0.04 and 0.03) patients. miR-146a and miR-21 expressions were significantly enhanced in UC patients compared to UC remission (UC-R; P = 0.01 and 0.04). Likewise, active CC patients showed significantly increased expression of miR-155 (P = 0.003) and miR-21 (P = 0.006). IL-37 expression was decreased in both CC (P = 0.03) and LC (P = 0.04) patients with a similar trend in UC patients but not statistically significant, whilst it was increased in UC-R patients compared to controls (P = 0.02) and active UC (P = 0.001). The identification of differentially expressed miRNAs, IL-37, and IRAK-M suggests different pathophysiologic mechanisms in various disease stages in LC, CC, and UC.

  16. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

    Science.gov (United States)

    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  17. μ-Opioid Agonist Inhibition of κ-Opioid Receptor-Stimulated Extracellular Signal-Regulated Kinase Phosphorylation Is Dynamin-Dependent in C6 Glioma Cells

    OpenAIRE

    Bohn, Laura M.; Belcheva, Mariana M.; Coscia, Carmine J.

    2000-01-01

    In previous studies we found that μ-opioids, acting via μ-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the κ-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with μ-opioid agonists for 1 h results in the inhibition of κ-opioid mitogenic signaling. The μ-selective agonist endomorphin-1 attenuates κ-opioid-stimu...

  18. Prelimbic cortex extracellular signal-regulated kinase 1/2 activation is required for memory retrieval of long-term inhibitory avoidance.

    Science.gov (United States)

    Luo, Fei; Zheng, Jian; Sun, Xuan; Deng, Wei-Ke; Li, Bao Ming; Liu, Fang

    2017-04-15

    Neural mechanism underlying memory retrieval has been extensively studied in the hippocampus and amygdala. However, little is known about the role of medial prefrontal cortex in long-term memory retrieval. We evaluate this issue in one-trial step-through inhibitory avoidance (IA) paradigm. Our results showed that, 1) inactivation of mPFC by local infusion of GABAA-receptor agonist muscimol caused severe deficits in retrieval of 1-day and 7-day but had no effects on 2-h inhibitory avoidance memory; 2) the protein level of phosphorylated-ERK1/2 in mPFC were significantly increased following retrieval of 1-day and 7-day IA memory, so did the numbers of phosphorylated-ERK (pERK) and phosphorylated-CREB (pCREB) labeled neurons; 3) intra-mPFC infusion of ERK kinase inhibitor PD98095 significantly reduced phosphorylated ERK1/2 levels and phosphorylated-ERK1/2 and phosphorylated-CREB labeled cells, and severely impaired retrieval of 7-day IA memory when the drugs were administrated 30min prior to test. The present study provides evidence that retrieval of long-lasting memory for inhibitory avoidance requires mPFC and involves the ERK-CREB signaling cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

    DEFF Research Database (Denmark)

    Hansen, Jakob Lerche; Aplin, Mark; Hansen, Jonas Tind;

    2008-01-01

    The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensi...... by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes....

  20. Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia

    Directory of Open Access Journals (Sweden)

    Kobayashi Kimiko

    2008-04-01

    Full Text Available Abstract Background Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK in the generation of BV-induced pain hypersensitivity. Results We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38 was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn. Conclusion The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.

  1. Integrated bioinformatics, computational and experimental methods to discover novel Raf/extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells.

    Science.gov (United States)

    Chen, Yin; Zheng, Yaxin; Jiang, Qinglin; Qin, Feifei; Zhang, Yonghui; Fu, Leilei; He, Gu

    2017-02-15

    Beginning with our previously reported ERK inhibitor BL-EI001, we found Raf1 to be an important regulator in the ERK interactive network, and then we designed and synthesized a novel series of Raf1/ERK dual inhibitors against human breast cancers through integrative computational, synthetic and biological screening methods. Moreover, we found that compound 9d suppressed the proliferation of breast cancer cell lines and induced cellular apoptosis via a mitochondrial pathway with only partial dependence on Raf1 and ERK. Our results suggest that an integrative method including in silico design, chemical synthesis, biological screening and bioinformatics analysis could be an attractive strategy for the discovery of multi-target inhibitors against breast cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Activated extracellular signal-regulated kinases are necessary and sufficient to initiate tubulogenesis in renal tubular MDCK strain I cell cysts.

    Science.gov (United States)

    Hellman, Nathan E; Greco, Andres J; Rogers, Katherine K; Kanchagar, Chitra; Balkovetz, Daniel F; Lipschutz, Joshua H

    2005-10-01

    A classic in vitro model of renal cyst and tubule formation utilizes the Madin-Darby canine kidney (MDCK) cell line, of which two strains exist. Most cyst and tubule formation studies that utilized MDCK cells have been performed with MDCK strain II cells. MDCK strain II cells form hollow cysts in a three-dimensional collagen matrix over 10 days and tubulate in response to hepatocyte growth factor, which increases levels of active (phosphorylated) ERK1/2. In this study, we demonstrate that MDCK strain I cells also form cysts when grown in a collagen matrix; however, MDCK strain I cell cysts spontaneously initiate the primary steps in tubulogenesis. Analysis of time-lapse microscopy of both MDCK strain I and strain II cell cysts during the initial stages of tubulogenesis demonstrates a highly dynamic process with cellular extensions and retractions occurring rapidly and continuously. MDCK strain I cell cysts can spontaneously initiate tubulogenesis mainly because of relatively higher levels of active ERK in MDCK strain I, compared with strain II, cells. The presence of either of two distinct inhibitors of ERK activation (UO126 and PD09059) prevents tubulogenesis from occurring spontaneously in MDCK strain I cell cysts and, in response to hepatocyte growth factor, in strain II cell cysts. The difference between MDCK strain I and strain II cell lines is likely explained by differing embryological origins, with strain I cells being of collecting duct, and hence ureteric bud, origin. Ureteric bud cells also have high levels of active ERK and spontaneously tubulate in our in vitro collagen gel system, with tubulogenesis inhibited by UO126 and PD09059. These results suggest that a seminal event in kidney development may be the activation of ERK in the mesonephric duct/ureteric bud cells destined to form the collecting tubules.

  3. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

    Science.gov (United States)

    Young, Christian D; Zimmerman, Lisa J; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B; Gatza, Michael L; Morrison, Meghan M; Moore, Preston D; Whitwell, Corbin A; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E; Silva, Grace O; Patel, Premal; Brantley-Sieders, Dana M; Levin, Maren; Horiates, Marina; Palma, Norma A; Wang, Kai; Stephens, Philip J; Perou, Charles M; Weaver, Alissa M; O'Shaughnessy, Joyce A; Chang, Jenny C; Park, Ben Ho; Liebler, Daniel C; Cook, Rebecca S; Arteaga, Carlos L

    2015-07-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer*

    Science.gov (United States)

    Young, Christian D.; Zimmerman, Lisa J.; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B.; Gatza, Michael L.; Morrison, Meghan M.; Moore, Preston D.; Whitwell, Corbin A.; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E.; Silva, Grace O.; Patel, Premal; Brantley-Sieders, Dana M.; Levin, Maren; Horiates, Marina; Palma, Norma A.; Wang, Kai; Stephens, Philip J.; Perou, Charles M.; Weaver, Alissa M.; O'Shaughnessy, Joyce A.; Chang, Jenny C.; Park, Ben Ho; Liebler, Daniel C.; Cook, Rebecca S.; Arteaga, Carlos L.

    2015-01-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. PMID:25953087

  5. Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations

    DEFF Research Database (Denmark)

    Holck, Susanne; Bonde, Jesper; Pedersen, Helle

    2016-01-01

    detectable increases in phosphorylation of ERK (pERK), we stained biopsies from 36 CRC patients with activating mutations in the BRAF gene (BRAFV600E: BRAF(m)), the KRAS gene (KRAS(m)) or in neither (BRAF/KRAS(n)) with this optimized method. Staining was scored in blind-coded specimens by two observers....... Staining of stromal cells was used as a positive control. BRAF(m) or KRAS(m) tumors did not show higher staining scores than BRAF/KRAS(n) tumors. Although BRAFV600E staining occurred in over 90% of cancer cells in all 9 BRAF(m) tumors, 3 only showed staining for pERK in less than 10% of cancer cell nuclei....... The same applied to 4 of the 14 KRAS(m) tumors. A phophorylation-insensitive antibody demonstrated that lack of pERK staining did not reflect defect expression of ERK1/2 protein. Thus, increased staining for pERK does not correlate to BRAF or KRAS mutations even with a highly optimized procedure. Further...

  6. Rosiglitazone attenuates the metalloprotease/anti-metalloprotease imbalance in emphysema induced by cigarette smoke: involvement of extracellular signal-regulated kinase and NFκB signaling

    Directory of Open Access Journals (Sweden)

    Hou G

    2015-04-01

    Full Text Available Gang Hou, Yan Yin, Dan Han, Qiu-yue Wang, Jian Kang Department of Respiratory Medicine, the First Hospital of China Medical University, Shenyang, People’s Republic of China Objective: We investigated how rosiglitazone attenuated cigarette smoke (CS-induced emphysema in a rat model. In particular, we focused on its possible effects on the imbalance between metalloprotease (MMP and anti-MMP activity, mitogen-activated protein kinase (MAPK phosphorylation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB signaling pathway over-activation.Methods: A total of 36 Wistar rats were divided into three groups (n=12 each: animals were exposed to CS for 12 weeks in the absence (the CS group or presence of 30 mg/kg rosiglitazone (the rosiglitazone-CS [RCS] group; a control group was treated with the rosiglitazone vehicle only, without any CS exposure. Histopathology of lung tissue in all groups was evaluated to grade severity of the disease. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ, MMP2, and MMP9 in lung tissue were determined and compared using Western blotting and immunohistochemistry. Activation of MAPKs, NFκB, and the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα phosphorylation in lung tissue was examined by Western blotting.Results: Emphysema-related pathology, based on inter-alveolar wall distance and alveolar density, was less severe in the RCS group than in the CS group. Compared with the CS group, levels of PPARγ were higher in the RCS group, and levels of MMP2 and MMP9 proteins were lower in the RCS rats. Levels of activated MAPKs and NFκB were also lower, while the IκBαphosphorylation was increased in the lung tissue of RCS rats.Conclusion: Our findings suggest that oral administration of rosiglitazone attenuates the metalloprotease activity induced by CS, and the underlying mechanism might involve the activation of signaling pathways dependent on MAPKs or NFκB. Our results further suggest that PPARγ contributes to the pathogenesis of emphysema as well as airway inflammation induced by CS. Keywords: emphysema, chronic obstructive pulmonary disease, matrix metalloprotease9, matrix metalloprotease2, PPAR, NFκB

  7. P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx

    DEFF Research Database (Denmark)

    Amstrup, Jan; Novak, Ivana

    2003-01-01

    mutants we show that the N-terminus is important in activation of ERKs, whereas deletion of the last 230 amino acids in the C-terminus did not effect ERK activation. On the other hand, Ca2+ entry was impaired in C-terminal but not in N-terminal mutants. In cell suspensions prepared from rat pancreas we...

  8. Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells.

    Science.gov (United States)

    Podar, Klaus; Mostoslavsky, Gustavo; Sattler, Martin; Tai, Yu-Tzu; Hayashi, Toshiaki; Catley, Laurence P; Hideshima, Teru; Mulligan, Richard C; Chauhan, Dharminder; Anderson, Kenneth C

    2004-05-14

    Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.

  9. SET9-Mediated Regulation of TGF-β Signaling Links Protein Methylation to Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    Maximilianos Elkouris

    2016-06-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  11. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guang; Li, Yan; Wang, Xiao-yu [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China); Han, Zhe [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Chuai, Manli [College of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH (United Kingdom); Wang, Li-jing [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Ho Lee, Kenneth Ka [Stem Cell and Regeneration Thematic Research Programme, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin (Hong Kong); Geng, Jian-guo, E-mail: jgeng@umich.edu [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109 (United States); Yang, Xuesong, E-mail: yang_xuesong@126.com [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China)

    2013-05-01

    development by tightly coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube.

  12. Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; Ramachandran, Anup [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Breckenridge, David G.; Liles, John T. [Department of Biology, Gilead Sciences, Inc., Foster City, CA (United States); Lebofsky, Margitta [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-07-01

    Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24 h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5 h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. - Highlights: • Two ASK1 inhibitors protected against acetaminophen-induced liver injury. • The ASK1 inhibitors protect when used as pre- or post-treatment. • Protection by ASK1 inhibitor is

  13. Extracellular proteolysis in the adult murine brain.

    Science.gov (United States)

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D

    1993-08-01

    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  14. Extracellular nucleotide signaling in plants

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  15. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    Science.gov (United States)

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  16. MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2011-12-01

    Full Text Available The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. Here we present the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels.

  17. NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity.

    Science.gov (United States)

    Baron, A; Montagne, A; Cassé, F; Launay, S; Maubert, E; Ali, C; Vivien, D

    2010-05-01

    Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

  18. Injury-Induced Type I IFN Signaling Regulates Inflammatory Responses in the Central Nervous System

    DEFF Research Database (Denmark)

    Khorooshi, Reza; Owens, Trevor

    2010-01-01

    Innate glial response is critical for the induction of inflammatory mediators and recruitment of leukocytes to sites of the injury in the CNS. We have examined the involvement of type I IFN signaling in the mouse hippocampus following sterile injury (transection of entorhinal afferents). Type I I...

  19. SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism

    NARCIS (Netherlands)

    Baster, P.; Robert, S.; Kleine-Vehn, J.; Vanneste, S.; Kania, U.; Grunewald, W.; Rybel, de B.P.M.; Beeckman, T.; Friml, J.

    2013-01-01

    The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels.

  20. SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism

    NARCIS (Netherlands)

    Baster, P.; Robert, S.; Kleine-Vehn, J.; Vanneste, S.; Kania, U.; Grunewald, W.; Rybel, de B.P.M.; Beeckman, T.; Friml, J.

    2013-01-01

    The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. Th

  1. Commensal-epithelial signaling mediated via formyl peptide receptors.

    Science.gov (United States)

    Wentworth, Christy C; Jones, Rheinallt M; Kwon, Young Man; Nusrat, Asma; Neish, Andrew S

    2010-12-01

    Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH(2)-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G(i)-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia.

  2. Extracellular Gd-CA

    DEFF Research Database (Denmark)

    Thomsen, Henrik S; Marckmann, Peter

    2008-01-01

    Until recently it was believed that extracellular gadolinium-based contrast agents were safe for both the kidneys and all other organs within the dose range up to 0.3 mmol/kg body weight. However, in 2006, it was demonstrated that some gadolinium-based contrast agents may trig the development of ...

  3. Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation.

    Science.gov (United States)

    Koole, Cassandra; Wootten, Denise; Simms, John; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M

    2012-02-03

    The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

  4. SmgGDS as a Crucial Mediator of the Inhibitory Effects of Statins on Cardiac Hypertrophy and Fibrosis: Novel Mechanism of the Pleiotropic Effects of Statins.

    Science.gov (United States)

    Kudo, Shun; Satoh, Kimio; Nogi, Masamichi; Suzuki, Kota; Sunamura, Shinichiro; Omura, Junichi; Kikuchi, Nobuhiro; Kurosawa, Ryo; Satoh, Taijyu; Minami, Tatsuro; Ikeda, Shohei; Miyata, Satoshi; Shimokawa, Hiroaki

    2016-05-01

    The detailed molecular mechanisms of the pleiotropic effects of statins remain to be fully elucidated. Here, we hypothesized that cardioprotective effects of statins are mediated by small GTP-binding protein GDP dissociation stimulator (SmgGDS). SmgGDS(+/-) and wild-type (WT) mice were treated with continuous infusion of angiotensin II (Ang II) for 2 weeks with and without oral treatment with atorvastatin or pravastatin. At 2 weeks, the extents of Ang II-induced cardiac hypertrophy and fibrosis were comparable between the 2 genotypes. However, statins significantly attenuated cardiomyocyte hypertrophy and fibrosis in WT mice, but not in SmgGDS(+/-) mice. In SmgGDS(+/-) cardiac fibroblasts (CFs), Rac1 expression, extracellular signal-regulated kinases 1/2 activity, Rho-kinase activity, and inflammatory cytokines secretion in response to Ang II were significantly increased when compared with WT CFs. Atorvastatin significantly reduced Rac1 expression and oxidative stress in WT CFs, but not in SmgGDS(+/-) CFs. Furthermore, Bio-plex analysis revealed significant upregulations of inflammatory cytokines/chemokines and growth factors in SmgGDS(+/-) CFs when compared with WT CFs. Importantly, conditioned medium from SmgGDS(+/-) CFs increased B-type natriuretic peptide expression in rat cardiomyocytes to a greater extent than that from WT CFs. Furthermore, atorvastatin significantly increased SmgGDS secretion from mouse CFs. Finally, treatment with recombinant SmgGDS significantly reduced Rac1 expression in SmgGDS(+/-) CFs. These results indicate that both intracellular and extracellular SmgGDS play crucial roles in the inhibitory effects of statins on cardiac hypertrophy and fibrosis, partly through inhibition of Rac1, Rho kinase, and extracellular signal-regulated kinase 1/2 pathways, demonstrating the novel mechanism of the pleiotropic effects of statins.

  5. A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Jagan Srinivasan

    2012-01-01

    Full Text Available The nematode C. elegans is an important model for the study of social behaviors. Recent investigations have shown that a family of small molecule signals, the ascarosides, controls population density sensing and mating behavior. However, despite extensive studies of C. elegans aggregation behaviors, no intraspecific signals promoting attraction or aggregation of wild-type hermaphrodites have been identified. Using comparative metabolomics, we show that the known ascarosides are accompanied by a series of derivatives featuring a tryptophan-derived indole moiety. Behavioral assays demonstrate that these indole ascarosides serve as potent intraspecific attraction and aggregation signals for hermaphrodites, in contrast to ascarosides lacking the indole group, which are repulsive. Hermaphrodite attraction to indole ascarosides depends on the ASK amphid sensory neurons. Downstream of the ASK sensory neuron, the interneuron AIA is required for mediating attraction to indole ascarosides instead of the RMG interneurons, which previous studies have shown to integrate attraction and aggregation signals from ASK and other sensory neurons. The role of the RMG interneuron in mediating aggregation and attraction is thought to depend on the neuropeptide Y-like receptor NPR-1, because solitary and social C. elegans strains are distinguished by different npr-1 variants. We show that indole ascarosides promote attraction and aggregation in both solitary and social C. elegans strains. The identification of indole ascarosides as aggregation signals reveals unexpected complexity of social signaling in C. elegans, which appears to be based on a modular library of ascarosides integrating building blocks derived from lipid β-oxidation and amino-acid metabolism. Variation of modules results in strongly altered signaling content, as addition of a tryptophan-derived indole unit to repellent ascarosides produces strongly attractive indole ascarosides. Our findings show

  6. A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans.

    Science.gov (United States)

    Srinivasan, Jagan; von Reuss, Stephan H; Bose, Neelanjan; Zaslaver, Alon; Mahanti, Parag; Ho, Margaret C; O'Doherty, Oran G; Edison, Arthur S; Sternberg, Paul W; Schroeder, Frank C

    2012-01-01

    The nematode C. elegans is an important model for the study of social behaviors. Recent investigations have shown that a family of small molecule signals, the ascarosides, controls population density sensing and mating behavior. However, despite extensive studies of C. elegans aggregation behaviors, no intraspecific signals promoting attraction or aggregation of wild-type hermaphrodites have been identified. Using comparative metabolomics, we show that the known ascarosides are accompanied by a series of derivatives featuring a tryptophan-derived indole moiety. Behavioral assays demonstrate that these indole ascarosides serve as potent intraspecific attraction and aggregation signals for hermaphrodites, in contrast to ascarosides lacking the indole group, which are repulsive. Hermaphrodite attraction to indole ascarosides depends on the ASK amphid sensory neurons. Downstream of the ASK sensory neuron, the interneuron AIA is required for mediating attraction to indole ascarosides instead of the RMG interneurons, which previous studies have shown to integrate attraction and aggregation signals from ASK and other sensory neurons. The role of the RMG interneuron in mediating aggregation and attraction is thought to depend on the neuropeptide Y-like receptor NPR-1, because solitary and social C. elegans strains are distinguished by different npr-1 variants. We show that indole ascarosides promote attraction and aggregation in both solitary and social C. elegans strains. The identification of indole ascarosides as aggregation signals reveals unexpected complexity of social signaling in C. elegans, which appears to be based on a modular library of ascarosides integrating building blocks derived from lipid β-oxidation and amino-acid metabolism. Variation of modules results in strongly altered signaling content, as addition of a tryptophan-derived indole unit to repellent ascarosides produces strongly attractive indole ascarosides. Our findings show that the library of

  7. α1-Adrenoceptor-mediated Ca2+-entry from the extracellular fluid and Ca2+-release from intracellular stores: No role for α(1A,B)-adrenoceptor subtypes in the pithed rat

    NARCIS (Netherlands)

    Schwietert, H.R.; Mathy, M.-J.; Wilhelm, D.; Wilffert, B.; Pfaffendorf, M.; Van Zwieten, P.A.

    1992-01-01

    1. In the present study, we tested the hypothesis that in the pithed rat preparation two subtypes of the α1-adrenoceptor are linked to two different signal transduction mechanisms, both of which contribute to vasoconstriction, one facilitating Ca2+-entry from the extracellular fluid (α(1A)) and one

  8. Ras signalling regulates differentiation and UCP1 expression in models of brown adipogenesis

    DEFF Research Database (Denmark)

    Murholm, Maria; Dixen, Karen; Hansen, Jacob B

    2010-01-01

    on two unrelated models of mouse brown adipocyte differentiation. RESULTS: A constitutively active H-Ras mutant (Ras V12) caused a complete block of adipose conversion, as manifested by a lack of both lipid accumulation and induction of adipocyte gene expression. The Ras V12-mediated impediment......-Ras mutant (Ras N17) did not inhibit differentiation, but led to increased expression of genes important for energy dissipation in brown fat cells, including UCP1. GENERAL SIGNIFICANCE: These data suggest that the intensity of Ras signalling is important for differentiation and UCP1 expression in models...

  9. FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans.

    Science.gov (United States)

    Minor, Paul J; He, Ting-Fang; Sohn, Chang Ho; Asthagiri, Anand R; Sternberg, Paul W

    2013-09-01

    The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

  10. ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis.

    Science.gov (United States)

    Shih, Hung Ping; Panlasigui, Devin; Cirulli, Vincenzo; Sander, Maike

    2016-01-12

    During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis

    Directory of Open Access Journals (Sweden)

    Hung Ping Shih

    2016-01-01

    Full Text Available During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

  12. Rho1-Wnd signaling regulates loss-of-cell polarity-induced cell invasion in Drosophila.

    Science.gov (United States)

    Ma, X; Chen, Y; Zhang, S; Xu, W; Shao, Y; Yang, Y; Li, W; Li, M; Xue, L

    2016-02-18

    Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. Here we show results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identify Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. We show that Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with Ras(V12) to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, our data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.

  13. Stat3 signaling regulates embryonic stem cell fate in a dose-dependent manner

    Directory of Open Access Journals (Sweden)

    Chih-I Tai

    2014-09-01

    Full Text Available Stat3 is essential for mouse embryonic stem cell (mESC self-renewal mediated by LIF/gp130 receptor signaling. Current understanding of Stat3-mediated ESC self-renewal mechanisms is very limited, and has heretofore been dominated by the view that Stat3 signaling functions in a binary “on/off” manner. Here, in contrast to this binary viewpoint, we demonstrate a contextual, rheostat-like mechanism for Stat3's function in mESCs. Activation and expression levels determine whether Stat3 functions in a self-renewal or a differentiation role in mESCs. We also show that Stat3 induces rapid differentiation of mESCs toward the trophectoderm (TE lineage when its activation level exceeds certain thresholds. Stat3 induces this differentiation phenotype via induction of Tfap2c and its downstream target Cdx2. Our findings provide a novel concept in the realm of Stat3, self-renewal signaling, and pluripotent stem cell biology. Ultimately, this finding may facilitate the development of conditions for the establishment of authentic non-rodent ESCs.

  14. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  15. Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes.

    Science.gov (United States)

    Booker, Matthew A; DeLong, Alison

    2015-09-01

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

  16. Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear.

    Science.gov (United States)

    Zhu, Xiao-Na; Liu, Xian-Dong; Zhuang, Hanyi; Henkemeyer, Mark; Yang, Jing-Yu; Xu, Nan-Jie

    2016-09-28

    The amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to environmental aversive cues. However, the molecular mechanism underlying the initial neuron responses is poorly understood. In this study, we monitored the innate defensive responses to aversive stimuli of either elevated plus maze or predator odor in juvenile mice and found that glutamatergic neurons were activated in amygdala. Loss of EphB2, a receptor tyrosine kinase expressed in amygdala neurons, suppressed the reactions and led to defects in spine morphogenesis and fear behaviors. We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2. A constitutively active form of EphB2 was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3, a brain-enriched ligand to EphB2. These data suggest that kinase-dependent EphB2 intracellular signaling plays a major role for innate fear responses during the critical developing period, in which spinogenesis in amygdala glutamatergic neurons was involved. Generation of innate fear responses to threat as an evolutionally conserved brain feature relies on development of functional neural circuit in amygdala, but the molecular mechanism remains largely unknown. We here identify that EphB2 receptor tyrosine kinase, which is specifically expressed in glutamatergic neurons, is required for the innate fear responses in the neonatal brain. We further reveal that EphB2 mediates coordination of spinogenesis and neuron activation in amygdala during the critical period for the innate fear. EphB2 catalytic activity plays a major role for the behavior upon EphB-ephrin-B3 binding and transnucleus neuronal connections. Our work thus indicates an essential synaptic molecular signaling within amygdala that controls synapse development and helps bring about innate fear emotions in the postnatal

  17. TOR signaling regulates planarian stem cells and controls localized and organismal growth.

    Science.gov (United States)

    Peiris, T Harshani; Weckerle, Frank; Ozamoto, Elyse; Ramirez, Daniel; Davidian, Devon; García-Ojeda, Marcos E; Oviedo, Néstor J

    2012-04-01

    Target of Rapamycin (TOR) controls an evolutionarily conserved signaling pathway that modulates cellular growth and division by sensing levels of nutrients, energy and stress. As such, TOR signaling is a crucial component of tissues and organs that translates systemic signals into cellular behavior. The ubiquitous nature of TOR signaling, together with the difficulty of analyzing tissue during cellular turnover and repair, have limited our understanding of how this kinase operates throughout the body. Here, we use the planarian model system to address TOR regulation at the organismal level. The planarian TOR homolog (Smed-TOR) is ubiquitously expressed, including stem cells (neoblasts) and differentiated tissues. Inhibition of TOR with RNA interference severely restricts cell proliferation, allowing the study of neoblasts with restricted proliferative capacity during regeneration and systemic cell turnover. Strikingly, TOR signaling is required for neoblast response to amputation and localized growth (blastema). However, in the absence of TOR signaling, regeneration takes place only within differentiated tissues. In addition, TOR is essential for maintaining the balance between cell division and cell death, and its dysfunction leads to tissue degeneration and lack of organismal growth in the presence of nutrients. Finally, TOR function is likely to be mediated through TOR Complex 1 as its disruption recapitulates signs of the TOR phenotype. Our data reveal novel roles for TOR signaling in controlling adult stem cells at a systemic level and suggest a new paradigm for studying TOR function during physiological turnover and regeneration.

  18. TDIF peptide signaling regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis.

    Science.gov (United States)

    Hirakawa, Yuki; Kondo, Yuki; Fukuda, Hiroo

    2010-08-01

    The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell-cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.

  19. Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis.

    Science.gov (United States)

    Ren, Maozhi; Venglat, Prakash; Qiu, Shuqing; Feng, Li; Cao, Yongguo; Wang, Edwin; Xiang, Daoquan; Wang, Jinghe; Alexander, Danny; Chalivendra, Subbaiah; Logan, David; Mattoo, Autar; Selvaraj, Gopalan; Datla, Raju

    2012-12-01

    Target of Rapamycin (TOR) is a major nutrition and energy sensor that regulates growth and life span in yeast and animals. In plants, growth and life span are intertwined not only with nutrient acquisition from the soil and nutrition generation via photosynthesis but also with their unique modes of development and differentiation. How TOR functions in these processes has not yet been determined. To gain further insights, rapamycin-sensitive transgenic Arabidopsis thaliana lines (BP12) expressing yeast FK506 Binding Protein12 were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf, and shoot growth and development leading to poor nutrient uptake and light energy utilization. Experimental limitation of nutrient availability and light energy supply in wild-type Arabidopsis produced phenotypes observed with TOR knockdown plants, indicating a link between TOR signaling and nutrition/light energy status. Genetic and physiological studies together with RNA sequencing and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and life span in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, and rRNA and protein synthesis. Gain- and loss-of-function Ribosomal Protein S6 (RPS6) mutants additionally show that TOR function involves RPS6-mediated nutrition and light-dependent growth and life span in Arabidopsis.

  20. CXCR2 signaling regulates KRAS(G12D)-induced autocrine growth of pancreatic cancer

    Science.gov (United States)

    Purohit, Abhilasha; Varney, Michelle; Rachagani, Satyanarayana; Ouellette, Michel M.; Batra, Surinder K.; Singh, Rakesh K.

    2016-01-01

    Pharmacological inhibition of RAS, the master regulator of pancreatic ductal adenocarcinoma (PDAC), continues to be a challenge. Mutations in various isoforms of RAS gene, including KRAS are known to upregulate CXC chemokines; however, their precise role in KRAS-driven pancreatic cancer remains unclear. In this report, we reveal a previously unidentified tumor cell-autonomous role of KRAS(G12D)-induced CXCR2 signaling in mediating growth of neoplastic PDAC cells. Progressively increasing expression of mCXCR2 and its ligands was detected in the malignant ductal cells of Pdx1-cre;LSL-Kras(G12D) mice. Knocking-down CXCR2 in KRAS(G12D)-bearing human pancreatic duct-derived cells demonstrated a significant decrease in the in vitro and in vivo tumor cell proliferation. Furthermore, CXCR2 antagonists showed selective growth inhibition of KRAS(G12D)-bearing cells in vitro. Intriguingly, both genetic and pharmacological inhibition of CXCR2 signaling in KRAS(G12D)-bearing pancreatic ductal cells reduced the levels of KRAS protein, strongly implying the presence of a KRAS-CXCR2 feed-forward loop. Together, these data demonstrate the role of CXCR2 signaling in KRAS(G12D)-induced growth transformation and progression in PDAC. PMID:26771140

  1. Macrophage Wnt-Calcineurin-Flt1 signaling regulates mouse wound angiogenesis and repair.

    Science.gov (United States)

    Stefater, James A; Rao, Sujata; Bezold, Katie; Aplin, Alfred C; Nicosia, Roberto F; Pollard, Jeffrey W; Ferrara, Napoleone; Lang, Richard A

    2013-03-28

    The treatment of festering wounds is one of the most important aspects of medical care. Macrophages are important components of wound repair, both in fending off infection and in coordinating tissue repair. Here we show that macrophages use a Wnt-Calcineurin-Flt1 signaling pathway to suppress wound vasculature and delay repair. Conditional mutants deficient in both Wntless/GPR177, the secretory transporter of Wnt ligands, and CNB1, the essential component of the nuclear factor of activated T cells dephosporylation complex, displayed enhanced angiogenesis and accelerated repair. Furthermore, in myeloid-like cells, we show that noncanonical Wnt activates Flt1, a naturally occurring inhibitor of vascular endothelial growth factor-A-mediated angiogenesis, but only when calcineurin function is intact. Then, as expected, conditional deletion of Flt1 in macrophages resulted in enhanced wound angiogenesis and repair. These results are consistent with the published link between enhanced angiogenesis and enhanced repair, and establish novel therapeutic approaches for treatment of wounds.

  2. Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth.

    Science.gov (United States)

    Chan, Audrey S M; Clairfeuille, Thomas; Landao-Bassonga, Euphemie; Kinna, Genevieve; Ng, Pei Ying; Loo, Li Shen; Cheng, Tak Sum; Zheng, Minghao; Hong, Wanjin; Teasdale, Rohan D; Collins, Brett M; Pavlos, Nathan J

    2016-04-15

    The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that sorting nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development. © 2016 Chan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  3. Guidance signalling regulates leading edge behaviour during collective cell migration of cardiac cells in Drosophila.

    Science.gov (United States)

    Raza, Qanber; Jacobs, J Roger

    2016-11-15

    Collective cell migration is the coordinated movement of cells, which organize tissues during morphogenesis, repair and some cancers. The motile cell membrane of the advancing front in collective cell migration is termed the Leading Edge. The embryonic development of the vertebrate and Drosophila hearts are both characterized by the coordinated medial migration of a bilateral cluster of mesodermal cells. In Drosophila, the cardioblasts form cohesive bilateral rows that migrate collectively as a unit towards the dorsal midline to form the dorsal vessel. We have characterized the collective cell migration of cardioblasts as an in vivo quantitative model to study the behaviour of the Leading Edge. We investigated whether guidance signalling through Slit and Netrin pathways plays a role in cell migration during heart development. Through time-lapse imaging and quantitative assessment of migratory behaviour of the cardioblasts in loss-of-function mutants, we demonstrate that both Slit and Netrin mediated signals are autonomously and concomitantly required to maximize migration velocity, filopodial and lamellipodial activities. Additionally, we show that another Slit and Netrin receptor, Dscam1, the role of which during heart development was previously unknown, is required for both normal migration of cardioblasts and luminal expansion. Leading edge behaviour analysis revealed a dosage dependent genetic interaction between Slit and Netrin receptors suggesting that downstream signalling through these receptors converge on a common output that increases leading edge activity of the cardioblasts. Finally, we found that guidance signalling maintains the balance between epithelial and mesenchymal characteristics of the migrating cardioblasts.

  4. Macroglia-Microglia Interactions via TSPO Signaling Regulates Microglial Activation in the Mouse Retina

    Science.gov (United States)

    Wang, Minhua; Wang, Xu; Zhao, Lian; Ma, Wenxin; Rodriguez, Ignacio R.; Fariss, Robert N.

    2014-01-01

    Chronic retinal inflammation in the form of activated microglia and macrophages are implicated in the etiology of neurodegenerative diseases of the retina, including age-related macular degeneration, diabetic retinopathy, and glaucoma. However, molecular biomarkers and targeted therapies for immune cell activation in these disorders are currently lacking. To address this, we investigated the involvement and role of translocator protein (TSPO), a biomarker of microglial and astrocyte gliosis in brain degeneration, in the context of retinal inflammation. Here, we find that TSPO is acutely and specifically upregulated in retinal microglia in separate mouse models of retinal inflammation and injury. Concomitantly, its endogenous ligand, diazepam-binding inhibitor (DBI), is upregulated in the macroglia of the mouse retina such as astrocytes and Müller cells. In addition, we discover that TSPO-mediated signaling in microglia via DBI-derived ligands negatively regulates features of microglial activation, including reactive oxygen species production, TNF-α expression and secretion, and microglial proliferation. The inducibility and effects of DBI-TSPO signaling in the retina reveal a mechanism of coordinated macroglia-microglia interactions, the function of which is to limit the magnitude of inflammatory responses after their initiation, facilitating a return to baseline quiescence. Our results indicate that TSPO is a promising molecular marker for imaging inflammatory cell activation in the retina and highlight DBI-TSPO signaling as a potential target for immodulatory therapies. PMID:24599476

  5. β-Catenin signaling regulates temporally discrete phases of anterior taste bud development

    Science.gov (United States)

    Thirumangalathu, Shoba; Barlow, Linda A.

    2015-01-01

    The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh+ placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh+ precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. PMID:26525674

  6. Multiple Mobile mRNA Signals Regulate Tuber Development in Potato

    Directory of Open Access Journals (Sweden)

    David J. Hannapel

    2017-02-01

    Full Text Available Included among the many signals that traffic through the sieve element system are full-length mRNAs that function to respond to the environment and to regulate development. In potato, several mRNAs that encode transcription factors from the three-amino-loop-extension (TALE superfamily move from leaves to roots and stolons via the phloem to control growth and signal the onset of tuber formation. This RNA transport is enhanced by short-day conditions and is facilitated by RNA-binding proteins from the polypyrimidine tract-binding family of proteins. Regulation of growth is mediated by three mobile mRNAs that arise from vasculature in the leaf. One mRNA, StBEL5, functions to activate growth, whereas two other, sequence-related StBEL’s, StBEL11 and StBEL29, function antagonistically to repress StBEL5 target genes involved in promoting tuber development. This dynamic system utilizes closely-linked phloem-mobile mRNAs to control growth in developing potato tubers. In creating a complex signaling pathway, potato has evolved a long-distance transport system that regulates underground organ development through closely-associated, full-length mRNAs that function as either activators or repressors.

  7. Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer.

    Science.gov (United States)

    Mori, Masaki; Triboulet, Robinson; Mohseni, Morvarid; Schlegelmilch, Karin; Shrestha, Kriti; Camargo, Fernando D; Gregory, Richard I

    2014-02-27

    Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here, we show that YAP, the downstream target of the tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding posttranscriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.

  8. Signaling regulating inner ear development: cell fate determination, patterning, morphogenesis, and defects.

    Science.gov (United States)

    Nakajima, Yuji

    2015-02-01

    The membranous labyrinth of the inner ear is a highly complex organ that detects sound and balance. Developmental defects in the inner ear cause congenital hearing loss and balance disorders. The membranous labyrinth consists of three semicircular ducts, the utricle, saccule, and endolymphatic ducts, and the cochlear duct. These complex structures develop from the simple otic placode, which is established in the cranial ectoderm adjacent to the neural crest at the level of the hindbrain at the early neurula stage. During development, the otic placode invaginates to form the otic vesicle, which subsequently gives rise to neurons for the vestibulocochlear ganglion, the non-sensory and sensory epithelia of the membranous labyrinth that includes three ampullary crests, two maculae, and the organ of Corti. Combined paracrine and autocrine signals including fibroblast growth factor, Wnt, retinoic acid, hedgehog, and bone morphogenetic protein regulate fate determination, axis formation, and morphogenesis in the developing inner ear. Juxtacrine signals mediated by Notch pathways play a role in establishing the sensory epithelium, which consists of mechanosensory hair cells and supporting cells. The highly differentiated organ of Corti, which consists of uniformly oriented inner/outer hair cells and specific supporting cells, develops during fetal development. Developmental alterations/arrest causes congenital malformations in the inner ear in a spatiotemporal-restricted manner. A clearer understanding of the mechanisms underlying inner ear development is important not only for the management of patients with congenital inner ear malformations, but also for the development of regenerative therapy for impaired function.

  9. Vitamin D signaling regulates oral keratinocyte proliferation in vitro and in vivo.

    Science.gov (United States)

    Yuan, Feng-Ning F; Valiyaparambil, Jayasanker; Woods, Michael C; Tran, Huy; Pant, Rima; Adams, John S; Mallya, Sanjay M

    2014-05-01

    The secosteroidal hormone 1,25-dihyroxyvitamin D [1,25(OH)(2)D(3)] and its receptor, the vitamin D receptor (VDR), are crucial regulators of epidermal proliferation and differentiation. However, the effects of 1,25(OH)(2)D(3)-directed signaling on oral keratinocyte pathophysiology have not been well studied. We examined the role of 1,25(OH)(2)D(3) in regulating proliferation and differentiation in cultured oral keratinocytes and on the oral epithelium in vivo. Using lentiviral-mediated shRNA to silence VDR, we generated an oral keratinocyte cell line with stable knockdown of VDR expression. VDR knockdown significantly enhanced proliferation and disrupted calcium- and 1,25(OH)(2)D(3)-induced oral keratinocyte differentiation, emphasizing the anti-proliferative and pro-differentiation effects of 1,25(OH)(2)D(3) in oral keratinocytes. Using vitamin D(3)-deficient diets, we induced chronic vitamin D deficiency in mice as evidenced by decreased serum 25-hydroxyvitamin D (25OHD) concentrations. The vitamin D-deficient mice manifested increased proliferation of the tongue epithelium, but did not develop any morphological or histological abnormalities in the oral epithelium, suggesting that vitamin D deficiency alone is insufficient to alter oral epithelial homeostasis and provoke carcinogenesis. Immunohistochemical analyses of human and murine oral squamous cell carcinomas showed increased VDR expression. Overall, our results provide strong support for a crucial role for vitamin D signaling in oral keratinocyte pathophysiology.

  10. A conserved juxtacrine signal regulates synaptic partner recognition in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Mock Natalyn

    2011-06-01

    Full Text Available Abstract Background An essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR. To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans. Results Here, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response

  11. Ets-1 upregulation mediates angiotensin II-related cardiac fibrosis.

    Science.gov (United States)

    Hao, Guanghua; Han, Zhenhua; Meng, Zhe; Wei, Jin; Gao, Dengfeng; Zhang, Hong; Wang, Nanping

    2015-01-01

    Ets-1, the prototypical member of the family of Ets transcription factors, has been shown to participate in tissue fibrotic remodeling. However, its role in cardiac fibrosis has not been established. The aim of this study was to investigate the role of Ets-1 in profibrotic actions of angiotensin II (Ang II) in cardiac fibroblasts (CFs) and in the in vivo heart. In growth-arrested CFs, Ang II induced Ets-1 expression in a time- and concentration-dependent manner. Pretreatment with Ang II type 1 receptor blocker losartan, protein kinase C inhibitor bisindolylmaleimide I, extracellular signal-regulated kinase (ERK) inhibitor PD98059, or c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 partly inhibited this induction accompanied with impaired cell proliferation and production of plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) protein, the two downstream targets of Ets-1. Knockdown of Ets-1 by siRNA significantly inhibited the inductive effects of Ang II on cell proliferation and expression of CTGF and PAI-1. Moreover, the levels of Ets-1, PAI-1 and CTGF protein were simultaneously upregulated in left ventricle of Ang II-infused rats in parallel with an increase in the activation of ERK and JNK. Our data suggest that Ets-1 may mediate Ang II-induced cardiac fibrotic effects.

  12. Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Larsen, Lise;

    2012-01-01

    component of extracellular matrix (ECM) in plaques, and to establish whether the pro-inflammatory molecules, tumor necrosis factor (TNF)-alpha, and receptor activator of nuclear factor-κB ligand (RANK-L) increase this degradation. CD14+ monocytes isolated from peripheral blood were differentiated......% and 72%, respectively. This is, to our knowledge, the first data describing a simple in vitro system in which macrophage foam cells degradation of matrix proteins can be monitored. This degradation can be enhanced by cytokines since TNF-alpha and RANK-L significantly increased the matrix degradation...

  13. Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

    Directory of Open Access Journals (Sweden)

    Deanna M Navaroli

    Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

  14. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases.

    Directory of Open Access Journals (Sweden)

    Andrea Seper

    Full Text Available The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

  15. Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

    Science.gov (United States)

    Gorgojo, Juan; Scharrig, Emilia; Gómez, Ricardo M.; Harvill, Eric T.; Rodríguez, Maria Eugenia

    2017-01-01

    B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts. PMID:28095485

  16. Extracellular conversion of adiponectin hexamers into trimers

    Science.gov (United States)

    Kim, Jeong-a; Nuñez, Martha; Briggs, David B.; Laskowski, Bethany L.; Chhun, Jimmy J.; Eleid, Joseph K.; Quon, Michael J.; Tsao, Tsu-Shuen

    2012-01-01

    Adiponectin is an adipocyte-secreted hormone that exists as trimers, hexamers and larger species collectively referred to as HMW (high-molecular-weight) adiponectin. Whether hexamers or HMW adiponectin serve as precursors for trimers outside the circulation is currently unknown. Here, we demonstrate that adiponectin trimers can be generated from larger oligomers secreted from primary rat adipose cells or differentiated 3T3-L1 adipocytes. Purified hexameric, but not HMW, adiponectin converted into trimers in conditioned media separated from 3T3-L1 adipocytes or, more efficiently, when enclosed in the dialysis membrane in the presence of adipocytes. Several lines of evidence indicate that the conversion is mediated by an extracellular redox system. First, N-terminal epitope-tagged hexamers converted into trimers without proteolytic removal of the tag. Secondly, appearance of trimers was associated with conversion of disulfide-bonded dimers into monomers. Thirdly, thiol-reactive agents inhibited conversion into trimers. Consistent with a redox-based mechanism, purified hexamers reductively converted into trimers in defined glutathione redox buffer with reduction potential typically found in the extracellular environment while the HMW adiponectin remained stable. In addition, conversion of hexamers into trimers was enhanced by NADPH, but not by NADP+. Collectively, these data strongly suggest the presence of an extracellular redox system capable of converting adiponectin oligomers. PMID:22973892

  17. Extracellular matrix proteins involved in pseudoislets formation.

    Science.gov (United States)

    Maillard, Elisa; Sencier, Marie-Christine; Langlois, A; Bietiger, William; Krafft, Mp; Pinget, Michel; Sigrist, Séverine

    2009-01-01

    Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

  18. Epithelial adhesion mediated by pilin SpaC is required for Lactobacillus rhamnosus GG-induced cellular responses.

    Science.gov (United States)

    Ardita, Courtney S; Mercante, Jeffrey W; Kwon, Young Man; Luo, Liping; Crawford, Madelyn E; Powell, Domonica N; Jones, Rheinallt M; Neish, Andrew S

    2014-08-01

    Lactobacillus rhamnosus GG is a widely used probiotic, and the strain's salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG's probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GGΩspaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG's capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases.

  19. Involvement of Nrf2-mediated heme oxygenase-1 expression in anti-inflammatory action of chitosan oligosaccharides through MAPK activation in murine macrophages.

    Science.gov (United States)

    Hyung, Jun-Ho; Ahn, Chang-Bum; Il Kim, Boo; Kim, Ky