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Sample records for induces creb phosphorylation

  1. Podophyllotoxin induces CREB phosphorylation and CRE-driven gene expression via PKA but not MAPKs.

    PubMed

    Chen, Ya Qiong; Xie, Xin

    2010-01-01

    CRE-driven luciferase reporter is commonly used in drug screening systems involving G protein-coupled receptors (GPCRs). In a screen campaign designed to search for melanocortin-4 receptor (MC4R) agonists, podophyllotoxin, a microtubules disruptor, was found to induce cAMP-responsive element (CRE)-driven reporter expression. MC4R was not involved because podophyllotoxin induced CREB activation and CRE-driven transcription in cells not expressing MC4R. Previous studies indicated that intracellular calcium, PKA, and MAPKs are involved in CREB phosphorylation and activation. Our studies revealed that podophyllotoxin did not affect intracellular calcium level and the phosphorylation state of p38. Podophyllotoxin induced JNK and ERK activation, but blockade of JNK and ERK activation with specific inhibitors had no effect on podophyllotoxin-induced CREB activation and CRE-regulated gene expression. Further experiments revealed that H89, a specific inhibitor of PKA, significantly inhibited podophyllotoxin-induced CREB activation. Podophyllotoxin itself did not alter intracellular cAMP level. Taken together, podophyllotoxin induces CREB activation and CRE-driven gene expression via PKA activation by a cAMP-independent mechanism.

  2. Nicotine-conditioned place preference induced CREB phosphorylation and Fos expression in the adult rat brain.

    PubMed

    Pascual, Mariano M; Pastor, Veronica; Bernabeu, Ramon O

    2009-11-01

    Experimental evidence indicates that nicotine causes long-lasting changes in the brain associated with behavior. Although much has been learned about factors participating in this process, less is known concerning the mechanisms and brain areas involved in nicotine preference. The objective of this study is to examine the participation of brain structures during the development of nicotine-conditioned place preference (CPP). To identify brain regions activated in CPP, we have measured the levels of phosphorylated cyclic AMP response element binding protein (pCREB) and Fos protein using a behavioral CPP and conditioned place aversion (CPA) paradigms. Rats developed reliable and robust CPP and also CPA. During nicotine preference and reinstatement behaviors, a significant increase of both pCREB and Fos protein expression occurs in the nucleus accumbens (NAc) and ventral tegmental area (VTA) and also in the prefrontal cortex (PFC), dorsal striatum (DStr), amygdala, and hippocampus. These increases were abolished by the administration of mecamylamine or by a CPA protocol, showing a specific activation of pCREB in drug preference animals, mediated by nicotinic receptors. Specifically in the VTA, nicotine-induced preference and reinstatement of the preference caused the activation of dopaminergic and GABAergic cells in different proportions. The results indicate that the phosphorylation of CREB and expression of Fos protein, as indicators of neural activity, accompany the acquisition and maintenance of nicotine-induced CPP but not CPA in mesolimbic areas (NAc, VTA, PFC, and DStr) as well as in memory consolidation structures (hippocampus and amygdala) and nicotinic receptor are involved in this process. Taken together, these studies identify the brain regions where pCREB activity is essential for nicotine preference.

  3. Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells

    PubMed Central

    Barlow, Christy A.; Kitiphongspattana, Kajorn; Siddiqui, Nazli; Roe, Michael W.; Mossman, Brooke T.

    2008-01-01

    Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress. PMID:18392938

  4. Chronic morphine administration induces over-expression of aldolase C with reduction of CREB phosphorylation in the mouse hippocampus.

    PubMed

    Yang, Hai-Yu; Pu, Xiao-Ping

    2009-05-01

    In recent studies, alterations in the activity and expression of metabolic enzymes, such as those involved in glycolysis, have been detected in morphine-dependent patients and animals. Increasing evidence demonstrates that the hippocampus is an important brain region associated with morphine dependence, but the molecular events occurring in the hippocampus following chronic exposure to morphine are poorly understood. Aldolase C is the brain-specific isoform of fructose-1, 6-bisphosphate aldolase which is a glycolytic enzyme catalyzing reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways. Using Western blot and immunofluorescence assays, we found the expression of aldolase C was markedly increased in the mouse hippocampus following chronic morphine treatment. Naloxone pretreatment before morphine administration suppressed withdrawal jumping, weight loss, and overexpression of aldolase C. CREB is a transcription factor regulated through phosphorylation on Ser133, which is known to play a key role in the mechanism of morphine dependence. When detecting the expression of phosphorylated CREB (p-CREB) in the mouse hippocampus using Western blot and immunohistochemistry, we found CREB phosphorylation was clearly decreased following chronic morphine treatment. Interestingly, laser-confocal microscopy showed that overexpression of aldolase C in mouse hippocampal neurons was concomitant with the decreased immunoreactivity of p-CREB. The results suggest potential links between the morphine-induced alteration of aldolase C and the regulation of CREB phosphorylation, a possible mechanism of morphine dependence.

  5. Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

    PubMed

    Jacques, Flavia Jesus; Silva, Thayane Martins; da Silva, Flavia Emenegilda; Ornelas, Isis Moraes; Ventura, Ana Lucia Marques

    2017-03-24

    Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [(3)H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY241385 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [(3)H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [(3)H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca(2+)]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca

  6. Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

    SciTech Connect

    Kwon, Yong-Jun; Sun, Yuanjie; Kim, Nam-Ho; Huh, Sung-Oh

    2009-03-13

    Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.

  7. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

    PubMed Central

    Nichols, M; Weih, F; Schmid, W; DeVack, C; Kowenz-Leutz, E; Luckow, B; Boshart, M; Schütz, G

    1992-01-01

    Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation. Images PMID:1354612

  8. GHRP-6 induces CREB phosphorylation and growth hormone secretion via a protein kinase Csigma-dependent pathway in GH3 cells.

    PubMed

    Tian, Chunlei; Ye, Fei; Xu, Tongjiang; Wang, Sheng; Wang, Xiaodan; Wang, Heping; Wan, Feng; Lei, Ting

    2010-04-01

    This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein (CREB) and the underly mechanism. GH3 cells were cultured and subjected to different treatments as follows: GHRP-6, GHRP-6 plus GHRH, phorbol ester (PMA), an activator of PKC, alone or in combination with GHRP-6, Gö6983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCsigma-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay (ELISA). The expression of phosphor-CREB, PKCsigma, PKCtheta and phosphor-PKCsigma was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors (Gö6983, rottlerin) and knockdown of PKCsigma. PKCsigma could be activated by GHRP-6. It is concluded that PKC, especially PKCsigma, mediates CREB phosphorylation and GHRP-6-induced GH secretion.

  9. Nobiletin improves brain ischemia-induced learning and memory deficits through stimulation of CaMKII and CREB phosphorylation.

    PubMed

    Yamamoto, Yui; Shioda, Norifumi; Han, Feng; Moriguchi, Shigeki; Nakajima, Akira; Yokosuka, Akihito; Mimaki, Yoshihiro; Sashida, Yutaka; Yamakuni, Tohru; Ohizumi, Yasushi; Fukunaga, Kohji

    2009-10-27

    Decreased cerebral blood flow causes cognitive impairments and neuronal injury in the progressive age-related neurodegenerative disorders such as Alzheimer's disease (AD) and vascular dementia. In the present study, we for the first time found that nobiletin, a novel leading compound for AD therapy, improved cerebral ischemia-induced memory deficits in vivo. Treatment with 50 mg/kg of nobiletin (i.p.) for the consecutive 7 days before and after brain ischemia significantly inhibited delayed neuronal death in the hippocampal CA1 neurons in a 20-min bilateral common carotid arteries occlusion (BCCAO) ischemia. However, the contextual memory assessed by passive avoidance task was not improved. On the other hand, a 5-min BCCAO-induced contextual memory deficit was significantly improved by the nobiletin treatment. In the 5-min BCCAO mice, Western blot analysis evidently showed that the levels of synaptic proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), microtubule-associated protein 2 (MAP2) and glutamate receptor 1 (GluR1), significantly decreased in the hippocampal CA1 region. The nobiletin treatment prevented the reduction in CaMKII, MAP2 and GluR1 protein levels in the hippocampal CA1 region, accompanied by restoration of both ERK and CREB phosphorylation and CaMKII autophosphorylation. Consistent with the restored CaMKII and ERK phosphorylation, an electrophysiological study showed that the impaired hippocampal long-term potentiation (LTP) observed in the 5-min ischemic mice was significantly improved by the nobiletin treatment. These findings suggest that the activation of CaMKII and ERK signaling in part mediates improvement of ischemia-induced learning and memory deficits by nobiletin.

  10. CP-154,526 Modifies CREB Phosphorylation and Thioredoxin-1 Expression in the Dentate Gyrus following Morphine-Induced Conditioned Place Preference.

    PubMed

    García-Carmona, Juan-Antonio; Camejo, Daymi M; Almela, Pilar; Jiménez, Ana; Milanés, María-Victoria; Sevilla, Francisca; Laorden, María-Luisa

    2015-01-01

    Corticotropin-releasing factor (CRF) acts as neuro-regulator of the behavioral and emotional integration of environmental and endogenous stimuli associated with drug dependence. Thioredoxin-1 (Trx-1) is a functional protein controlling the redox status of several proteins, which is involved in addictive processes. In the present study, we have evaluated the role of CRF1 receptor (CRF1R) in the rewarding properties of morphine by using the conditioned place preference (CPP) paradigm. We also investigate the effects of the CRF1R antagonist, CP-154,526, on the morphine CPP-induced activation of CRF neurons, CREB phosphorylation and Trx expression in paraventricular nucleus (PVN) and dentate gyrus (DG) of the mice brain. CP-154,526 abolished the acquisition of morphine CPP and the increase of CRF/pCREB positive neurons in PVN. Moreover, this CRF1R antagonist prevented morphine-induced CRF-immunoreactive fibers in DG, as well as the increase in pCREB expression in both the PVN and DG. In addition, morphine exposure induced an increase in Trx-1 expression in DG without any alterations in PVN. We also observed that the majority of pCREB positive neurons in DG co-expressed Trx-1, suggesting that Trx-1 could activate CREB in the DG, a brain region involved in memory consolidation. Altogether, these results support the idea that CRF1R antagonist blocked Trx-1 expression and pCREB/Trx-1 co-localization, indicating a critical role of CRF, through CRF1R, in molecular changes involved in morphine associated behaviors.

  11. Serine 133 Phosphorylation Is Not Required for Hippocampal CREB-Mediated Transcription and Behavior

    ERIC Educational Resources Information Center

    Brian, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.; Blendy, Julie A.

    2015-01-01

    The cAMP response element (CRE)-binding protein, CREB, is a transcription factor whose activity in the brain is critical for long-term memory formation. Phosphorylation of Ser133 in the kinase-inducible domain (KID), that in turn leads to the recruitment of the transcriptional coactivator CREB-binding protein (CBP), is thought to mediate the…

  12. Serine 133 Phosphorylation Is Not Required for Hippocampal CREB-Mediated Transcription and Behavior

    ERIC Educational Resources Information Center

    Brian, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.; Blendy, Julie A.

    2015-01-01

    The cAMP response element (CRE)-binding protein, CREB, is a transcription factor whose activity in the brain is critical for long-term memory formation. Phosphorylation of Ser133 in the kinase-inducible domain (KID), that in turn leads to the recruitment of the transcriptional coactivator CREB-binding protein (CBP), is thought to mediate the…

  13. Alterations in the hippocampal phosphorylated CREB expression in drug state-dependent learning.

    PubMed

    Alijanpour, Sakineh; Rezayof, Ameneh; Sepehri, Houri; Delphi, Ladan

    2015-10-01

    The present study investigated the possible alterations of hippocampal CREB phosphorylation in drug state-dependent memory retrieval. One-trial step-down passive avoidance task was used to assess memory retrieval in adult male NMRI mice. Pre-training administration of ethanol (1g/kg, i.p.) induced amnesia. Pre-test administration of ethanol (1g/kg, i.p) or nicotine (0.7 mg/kg, s.c.) reversed ethanol-induced amnesia, indicating ethanol- or ethanol-nicotine induced state-dependent learning (STD). Using Western blot analysis, it was found that the p-CREB/CREB ratio in the hippocampus increased in the mice that showed successful memory retrieval as compared with untrained mice. In contrast, pre-training administration of ethanol (1g/kg, i.p.) decreased the hippocampal p-CREB/CREB ratio in comparison with the control group. The hippocampal p-CREB/CREB ratio enhanced in ethanol- and ethanol-nicotine induced STD. Moreover, memory impairment induced by pre-training administration of WIN (1 mg/kg, i.p.) improved in the animals that received pre-test administration of WIN (1 mg/kg, i.p.), ethanol (0.5 g/kg, i.p.) or nicotine (0.7 mg/kg, s.c.), suggesting a cross STD between the drugs. The p-CREB/CREB ratio in the hippocampus decreased in the of WIN-induced amnesia and STD groups in comparison with the control group. In addition, cross state-dependent learning between WIN and ethanol or nicotine was associated with the increase of the hippocampal p-CREB/CREB ratio. It can be concluded that phosphorylation of CREB in the hippocampus is a critical event underlying the interaction of co-administration of drugs on memory retrieval in passive avoidance learning. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

    PubMed

    Sotelo-Rivera, Israim; Cote-Vélez, Antonieta; Uribe, Rosa-María; Charli, Jean-Louis; Joseph-Bravo, Patricia

    2017-03-01

    Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

  15. Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation

    PubMed Central

    Hsu, Chih‐Kai; Lin, Chih‐Chung; Hsiao, Li‐Der

    2015-01-01

    Background and Purpose Sphingosine 1‐phosphate (S1P), an important inflammatory mediator, has been shown to regulate COX‐2 production and promote various cellular responses such as cell migration. Mevastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CoA), effectively inhibits inflammatory responses. However, the mechanisms underlying S1P‐evoked COX‐2‐dependent cell migration, which is modulated by mevastatin in human tracheal smooth muscle cells (HTSMCs) remain unclear. Experimental Approach The expression of COX‐2 was determined by Western blotting, real time‐PCR and promoter analyses. The signalling molecules were investigated by pretreatment with respective pharmacological inhibitors or transfection with siRNAs. The interaction between COX‐2 promoter and transcription factors was determined by chromatin immunoprecipitation assay. Finally, the effect of mevastatin on HTSMC migration and leukocyte counts in BAL fluid and COX‐2 expression induced by S1P was determined by a cell migration assay, cell counting and Western blot. Key Results S1P stimulated mTOR activation through the Nox2/ROS and PI3K/Akt pathways, which can further stimulate FoxO1 phosphorylation and translocation to the cytosol. We also found that S1P induced CREB activation and translocation via an mTOR‐independent signalling pathway. Finally, we showed that pretreatment with mevastatin markedly reduced S1P‐induced cell migration and COX‐2/PGE2 production via a PPARγ‐dependent signalling pathway. Conclusions and Implications Mevastatin attenuates the S1P‐induced increased expression of COX‐2 and cell migration via the regulation of FoxO1 and CREB phosphorylation and translocation by PPARγ in HTSMCs. Mevastatin could be beneficial for prevention of airway inflammation in the future. PMID:26359950

  16. Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain.

    PubMed

    Réus, Gislaine Z; Stringari, Roberto B; Ribeiro, Karine F; Ferraro, Ana K; Vitto, Marcelo F; Cesconetto, Patrícia; Souza, Claúdio T; Quevedo, João

    2011-08-01

    A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10mg/kg) and imipramine (10 and 20mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10mg/kg and ketamine at the dose of 5mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.

  17. Impairment of Memory Consolidation by Galanin Correlates with In-Vivo Inhibition of Both LTP and CREB Phosphorylation

    PubMed Central

    Kinney, Jefferson W.; Sanchez-Alavez, Manuel; Barr, Alasdair M.; Criado, Jose R.; Crawley, Jacqueline N.; Behrens, M. Margarita; Henriksen, Steven J.; Bartfai, Tamas

    2009-01-01

    Changes in the state of CREB phosphorylation and in LTP in the hippocampus have been associated with learning and memory. Here we show that galanin, the neuropeptide released in the hippocampal formation from cholinergic and noradrenergic fibers, that has been shown to produce impairments in memory consolidation in the Morris water maze task inhibits both LTP and CREB phosphorylation in the rat hippocampus in-vivo. While there are many transmitters regulating CREB phosphorylation none has been shown to suppress behaviorally-induced hippocampal CREB phosphorylation as potently as galanin. The in-vivo inhibition of dentate gyrus-LTP and of CREB phosphorylation by the agonist occupancy of GalR1 and GALR2-type galanin receptors provides strong in-vivo cellular and molecular correlates to galanin-induced learning deficits and designates galanin as a major regulator of the memory consolidation process. PMID:19531380

  18. Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on serine 133.

    PubMed Central

    Houglum, K; Lee, K S; Chojkier, M

    1997-01-01

    Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation of activated primary rat stellate cells was blocked by elevation of cAMP with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhibitor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-23187. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) activation, we tested whether CREB-PSer133 was essential for stellate cell quiescence. Nuclear extracts from quiescent, but not from activated, stellate cells contained CREB-PSer133. Moreover, the phosphorylation of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and either a constitutively active PKA or a constitutively active CAMK-II inhibited the proliferation of activated stellate cells. In contrast, expression of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, suggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle. Conversely, expression of a trans-dominant negative CREB-Ala 133 mutant (which competes with CREB/CREB-PSer133 for cognate DNA binding sites and presumably for protein interactions) induced a greater than fivefold entry into S-phase of quiescent stellate cells, compared with control cells expressing either beta-galactosidase or wt CREB, indicating that CREB-PSer133 may be indispensable for the quiescent stellate cell phenotype. This study suggests that PKA and CAMK-II play an essential role on stellate cell activation through the induction of CREB phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in

  19. Characterization and phosphorylation of CREB-like proteins in Aplysia central nervous system.

    PubMed

    Dash, P K; Moore, A N

    1996-07-01

    Studies in Aplysia californica indicate that cAMP-mediated gene expression is necessary for long-term facilitation, a correlate of long-term memory. It has been shown that blocking the expression of cAMP-inducible genes in sensory neurons impedes long-term facilitation without any effect on short-term facilitation. Specifically, blocking the binding of CREB-like proteins or inhibiting the expression of a cAMP-inducible gene, C[symbon: see text]EBP, impairs long-term facilitation. In this report, we show the presence of a family of CREB-like proteins in Aplysia CNS that specifically bind to the CRE sequence and cross-react with rat CREB antibodies. Similar to mammalian CREB proteins, Aplysia homologues interact with each other via leucine zipper domains. This interaction can be disrupted by peptides containing the CREB leucine zipper sequence. We demonstrate that a 43 kDa CREB-like protein present in CNS extracts can be phosphorylated in vitro by cAMP-dependent protein kinase A. Moreover, exposure of ganglia to serotonin (5-HT), a transmitter involved in long-term facilitation, increases the phosphorylation of this protein. This biochemical data further supports the involvement of CREB-like proteins in memory storage.

  20. Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus

    ERIC Educational Resources Information Center

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

  1. Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus

    ERIC Educational Resources Information Center

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

  2. Blockade of Cannabinoid CB1 receptor attenuates the acquisition of morphine-induced conditioned place preference along with a downregulation of ERK, CREB phosphorylation, and BDNF expression in the nucleus accumbens and hippocampus.

    PubMed

    Zhang, Jianbo; Wang, Na; Chen, Bo; Wang, Yi'nan; He, Jing; Cai, Xintong; Zhang, Hongbo; Wei, Shuguang; Li, Shengbin

    2016-09-06

    Cannabinoid CB1 receptor (CB1R) is highly expressed in the mesocorticolimbic system and associated with drug craving and relapse. Clinical trials suggest that CB1R antagonists may represent new therapies for drug addiction. However, the downstream signaling of CB1R is not fully elucidated. In the present study, we investigated the relationship between CB1R and the extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF) signaling in the nucleus accumbens (NAc) and hippocampus in morphine-induced conditioned place preference (CPP), which is used to assess the morphine-induced reward memory. The protein level of CB1R, ERK, CREB, and BDNF were detected by western blotting. Additionally, a CB1R antagonist, AM251, was used to study whether blockade of CB1R altered the CPP and above-mentioned molecules. We found an increase of CB1R expression in the NAc and hippocampus of the mice following morphine CPP, but not those after repeated morphine in home cage without context exposure (NO-CPP). Both morphine CPP and NO-CPP induced an upregulation of ERK, CREB phosphorylation and BDNF expression. Furthermore, pretreatment with AM251 before morphine attenuated the CPP acquisition and CB1R expression as well as the activation of ERK-CREB-BDNF cascade. Collectively, these findings demonstrate that (1) Repeated morphine with context exposures but not merely the pharmacological effects of morphine increased CB1R expression both in the NAc and hippocampus. (2) CB1R antagonist mediated blockade of ERK-CREB-BDNF signaling activation in the NAc and hippocampus may be an important mechanism underlying the attenuation of morphine CPP. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

    PubMed

    Maronde, E; Wicht, H; Taskén, K; Genieser, H G; Dehghani, F; Olcese, J; Korf, H W

    1999-10-01

    Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

  4. Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus

    PubMed Central

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees (Apis mellifera) we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees’ CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus. PMID:27084927

  5. Oxidative stress-induced CREB upregulation promotes DNA damage repair prior to neuronal cell death protection.

    PubMed

    Pregi, Nicolás; Belluscio, Laura María; Berardino, Bruno Gabriel; Castillo, Daniela Susana; Cánepa, Eduardo Tomás

    2017-01-01

    cAMP response element-binding (CREB) protein is a cellular transcription factor that mediates responses to different physiological and pathological signals. Using a model of human neuronal cells we demonstrate herein, that CREB is phosphorylated after oxidative stress induced by hydrogen peroxide. This phosphorylation is largely independent of PKA and of the canonical phosphoacceptor site at ser-133, and is accompanied by an upregulation of CREB expression at both mRNA and protein levels. In accordance with previous data, we show that CREB upregulation promotes cell survival and that its silencing results in an increment of apoptosis after oxidative stress. Interestingly, we also found that CREB promotes DNA repair after treatment with hydrogen peroxide. Using a cDNA microarray we found that CREB is responsible for the regulation of many genes involved in DNA repair and cell survival after oxidative injury. In summary, the neuroprotective effect mediated by CREB appears to follow three essential steps following oxidative injury. First, the upregulation of CREB expression that allows sufficient level of activated and phosphorylated protein is the primordial event that promotes the induction of genes of the DNA Damage Response. Then and when the DNA repair is effective, CREB induces detoxification and survival genes. This kinetics seems to be important to completely resolve oxidative-induced neuronal damages.

  6. Effect of delta9-tetrahydrocannabinol on phosphorylated CREB in rat cerebellum: an immunohistochemical study.

    PubMed

    Casu, Maria Antonietta; Pisu, Carla; Sanna, Angela; Tambaro, Simone; Spada, Gabriele Pinna; Mongeau, Raymond; Pani, Luca

    2005-06-28

    Several converging lines of evidence indicate that drugs of abuse may exert their long-term effects on the central nervous system by modulating signaling pathways controlling gene expression. Cannabinoids produce, beside locomotor effects, cognitive impairment through central CB1 cannabinoid receptors. Data clearly indicate that the cerebellum, an area enriched with CB1 receptors, has a role not only in motor function but also in cognition. This immunohistochemical study examines the effect of delta9-tetrahydrocannabinol (delta9-THC), the principal psychoactive component of marijuana, on the levels of phosphorylated CREB (p-CREB) in the rat cerebellum. Acute treatments with delta9-THC at doses of 5 or 10 mg/kg induced a significant increase of p-CREB in the granule cell layer of the cerebellum, an effect blocked by the CB1 receptor antagonist SR 141716A. Following chronic delta9-THC administration (10 mg/kg/day for 4 weeks), the density of p-CREB was markedly attenuated compared to controls, and this attenuation persisted 3 weeks after withdrawal from delta9-THC. These data provide evidence for the involvement of cerebellar granule cells in the adaptive changes occurring during acute and chronic delta9-THC exposure. This might be a mechanism by which delta9-THC interferes with motor and cognitive functions.

  7. CREB Regulates AChE-R-Induced Proliferation of Human Glioblastoma Cells1

    PubMed Central

    Perry, Chava; Sklan, Ella H; Soreq, Hermona

    2004-01-01

    Abstract The cyclic adenosine monophosphate (AMP) response element-binding protein, CREB, often modulates stress responses. Here, we report that CREB suppresses the glioblastoma proliferative effect of the stress-induced acetylcholinesterase variant, AChE-R. In human U87MG glioblastoma cells, AChE-R formed a triple complex with protein kinase C (PKC) ε and the scaffold protein RACK1, enhanced PKCε phosphorylation, and facilitated BrdU incorporation. Either overexpressed CREB, or antisense destruction of AChE-R mRNA, PKC, or protein kinase A (PKA) inhibitors—but not CREB combined with PKC inhibition suppressed—this proliferation, suggesting that CREB's repression of this process involves a PKC-mediated pathway, whereas impaired CREB regulation allows AChE-R-induced, PKA-mediated proliferation of glioblastoma tumors. PMID:15153340

  8. CREB regulates AChE-R-induced proliferation of human glioblastoma cells.

    PubMed

    Perry, Chava; Sklan, Ella H; Soreq, Hermona

    2004-01-01

    The cyclic adenosine monophosphate (AMP) response element-binding protein, CREB, often modulates stress responses. Here, we report that CREB suppresses the glioblastoma proliferative effect of the stress-induced acetylcholinesterase variant, AChE-R. In human U87MG glioblastoma cells, AChE-R formed a triple complex with protein kinase C (PKC) epsilon and the scaffold protein RACK1, enhanced PKCepsilon phosphorylation, and facilitated BrdU incorporation. Either overexpressed CREB, or antisense destruction of AChE-R mRNA, PKC, or protein kinase A (PKA) inhibitors-but not CREB combined with PKC inhibition suppressed-this proliferation, suggesting that CREB's repression of this process involves a PKC-mediated pathway, whereas impaired CREB regulation allows AChE-R-induced, PKA-mediated proliferation of glioblastoma tumors.

  9. Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascades.

    PubMed

    Tiraboschi, Ettore; Tardito, Daniela; Kasahara, Jiro; Moraschi, Stefania; Pruneri, Paolo; Gennarelli, Massimo; Racagni, Giorgio; Popoli, Maurizio

    2004-10-01

    Regulation of gene expression is purported as a major component in the long-term action of antidepressants. The transcription factor cAMP-response element-binding protein (CREB) is activated by chronic antidepressant treatments, although a number of studies reported different effects on CREB, depending on drug types used and brain areas investigated. Furthermore, little is known as to what signaling cascades are responsible for CREB activation, although cAMP-protein kinase A (PKA) cascade was suggested to be a central player. We investigated how different drugs (fluoxetine (FLX), desipramine (DMI), reboxetine (RBX)) affect CREB expression and phosphorylation of Ser(133) in the hippocampus and prefrontal/frontal cortex (PFCX). Acute treatments did not induce changes in these mechanisms. Chronic FLX increased nuclear phospho-CREB (pCREB) far more markedly than pronoradrenergic drugs, particularly in PFCX. We investigated the function of the main signaling cascades that were shown to phosphorylate and regulate CREB. PKA did not seem to account for the selective increase of pCREB induced by FLX. All drug treatments markedly increased the enzymatic activity of nuclear Ca2+/calmodulin (CaM) kinase IV (CaMKIV), a major neuronal CREB kinase, in PFCX. Activation of this kinase was due to increased phosphorylation of the activatory residue Thr196, with no major changes in the expression levels of alpha- and beta-CaM kinase kinase, enzymes that phosphorylate CaMKIV. Again in PFCX, FLX selectively increased the expression level of MAP kinases Erk1/2, without affecting their phosphorylation. Our results show that FLX exerts a more marked effect on CREB phosphorylation and suggest that CaMKIV and MAP kinase cascades are involved in this effect.

  10. Menin and PRMT5 suppress GLP1 receptor transcript and PKA-mediated phosphorylation of FOXO1 and CREB.

    PubMed

    Muhammad, Abdul Bari; Xing, Bowen; Liu, Chengyang; Naji, Ali; Ma, Xiaosong; Simmons, Rebecca A; Hua, Xianxin

    2017-03-07

    Menin is a scaffold protein that interacts with several epigenetic mediators to regulate gene transcription, and suppresses pancreatic beta cell proliferation. Tamoxifen inducible deletion of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes the protein menin, increases beta cell mass in multiple murine models of diabetes and ameliorates diabetes. Glucagon-like-peptide-1 (GLP1) is another key physiological modulator of beta cell mass and glucose homeostasis. However, it is not clearly understood whether menin crosstalks with GLP1 signaling. Here we show that menin and protein arginine methyltransferase 5 (PRMT5) suppress GLP1 receptor (GLP1R) transcript levels. Notably, a GLP1R agonist induces phosphorylation of forkhead box protein O1 (FOXO1) at S253, and the phosphorylation is mediated by protein kinase A (PKA). Interestingly, menin suppresses GLP1-induced and PKA-mediated phosphorylation of both FOXO1 and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), likely through a protein arginine methyltransferase. Menin mediated suppression of FOXO1 and CREB phosphorylation increases FOXO1 levels and suppresses CREB target genes, respectively. A small molecule menin inhibitor reverses menin-mediated suppression of both FOXO1 and CREB phosphorylation. In addition, ex vivo treatment of both mouse and human pancreatic islets with a menin inhibitor increases levels of proliferation marker Ki67. In conclusion, our results suggest that menin and PRMT5 suppress GLP1R transcript levels and PKA-mediated phosphorylation of FOXO1 and CREB, and a menin inhibitor may reverse this suppression to induce beta cell proliferation.

  11. Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

    PubMed Central

    Böer, Ulrike; Eglins, Julia; Krause, Doris; Schnell, Susanne; Schöfl, Christof; Knepel, Willhart

    2007-01-01

    The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium. PMID:17696880

  12. Phosphorylation of CREB and mechanical hyperalgesia is reversed by blockade of the cAMP pathway in a time-dependent manner after repeated intramuscular acid injections.

    PubMed

    Hoeger-Bement, Marie K; Sluka, Kathleen A

    2003-07-02

    Spinal activation of the cAMP pathway produces mechanical hyperalgesia, sensitizes nociceptive spinal neurons, and phosphorylates the transcription factor cAMP-responsive element binding protein (CREB), which initiates gene transcription. This study examined the role of the cAMP pathway in a model of chronic muscle pain by assessing associated behavioral changes and phosphorylation of CREB. Bilateral mechanical hyperalgesia of the paw was induced by administering two injections of acidic saline, 5 d apart, into the gastrocnemius muscle of male Sprague Dawley rats. Interestingly, the increases in immunoreactivity for CREB and phosphorylated CREB (p-CREB) in the spinal dorsal horn occur 24 hr, but not 1 week, after the second injection of acidic saline compared with pH 7.2 intramuscular injections. Spinal blockade of adenylate cyclase prevents the expected increase in p-CREB that occurs after intramuscular acid injection. The reversal of mechanical hyperalgesia by adenylate cyclase or protein kinase A inhibitors spinally follows a similar pattern with reversal at 24 hr, but not 1 week, compared with the vehicle controls. The p-CREB immunoreactivity in the superficial dorsal horn correlates with the mechanical withdrawal threshold such that increases in p-CREB are associated with decreases in threshold. Therefore, activation of the cAMP pathway in the spinal cord phosphorylates CREB and produces mechanical hyperalgesia associated with intramuscular acid injections. The mechanical hyperalgesia and phosphorylation of CREB depend on early activation of the cAMP pathway during the first 24 hr but are independent of the cAMP pathway by 1 week after intramuscular injection of acid.

  13. Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum.

    PubMed

    Pozzi, Laura; Håkansson, Kerstin; Usiello, Alessandro; Borgkvist, Anders; Lindskog, Maria; Greengard, Paul; Fisone, Gilberto

    2003-07-01

    The two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), are involved in the control of gene expression via phosphorylation and activation of the transcription factors cyclic AMP response element binding protein (CREB) and Elk-1. Here, we have examined the effect of haloperidol and clozapine, two anti-psychotic drugs, and eticlopride, a selective dopamine D2 receptor antagonist, on the state of phosphorylation of ERK1/2, CREB and Elk-1, in the mouse dorsal striatum. Administration of the typical anti-psychotic haloperidol stimulated the phosphorylation of ERK1/2, CREB and Elk-1. Virtually identical results were obtained using eticlopride. In contrast, the atypical anti-psychotic clozapine reduced ERK1/2, CREB and Elk-1 phosphorylation. This opposite regulation was specifically exerted by haloperidol and clozapine on ERK, CREB, and Elk-1 phosphorylation, as both anti-psychotic drugs increased the phosphorylation of the dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) at the cyclic AMP-dependent protein kinase (PKA) site. The activation of CREB and Elk-1 induced by haloperidol appeared to be achieved via different signalling pathways, as inhibition of ERK1/2 activation abolished the stimulation of Elk-1 phosphorylation without affecting CREB phosphorylation. This study shows that haloperidol and clozapine induce distinct patterns of phosphorylation in the dorsal striatum. The results provide a novel biochemical paradigm elucidating the molecular mechanisms underlying the distinct therapeutic actions of typical and atypical anti-psychotic agents.

  14. Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation

    PubMed Central

    Bender, R. A.; Lauterborn, J. C.; Gall, C. M.; Cariaga, W.; Baram, T. Z.

    2011-01-01

    Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network. PMID:11207803

  15. Cyclic-AMP-responsive transcriptional activation of CREB-327 involves interdependent phosphorylated subdomains.

    PubMed Central

    Lee, C Q; Yun, Y D; Hoeffler, J P; Habener, J F

    1990-01-01

    Cyclic AMP-regulated gene expression is mediated by specific phosphoproteins (CREBs) which bind to cAMP-responsive elements of gene promoters. By analyzing the transactivation activities and phosphorylations in vivo of deletion and point mutated chimeric fusion proteins of the placental CREB-327, in which the DNA-binding domain is replaced by the heterologous binding-domain of the yeast transcription factor GAL4, we localized the cAMP-responsive and phosphorylated domain to a minimal-essential sequence module of 46 amino acids (residues 92-137). This serine-rich, multiply-phosphorylated sequence consists of at least three interdependent subdomains required for transcriptional activation. Although phosphorylation of serine-119 by cyclic AMP-dependent protein kinase A is necessary for transcriptional activation, such activation requires both a phosphorylated heptadecapeptide domain located ten residues amino terminal to the serine-119 and an eleven-residue domain carboxyl terminal to the serine-119. Deletion of these two domains does not impair phosphorylation of serine-119. Further, deletion of the carboxyl-terminal domain does not alter phosphorylation of the heptadecapeptide domain. We propose that akin to the phosphorylation-dependent activation of enzymes, the transcriptional transactivation functions of CREB-327 involve a phosphorylation-dependent allosteric conformational mechanism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2176153

  16. Cadmium up-regulates transcription of the steroidogenic acute regulatory protein (StAR) gene through phosphorylated CREB rather than SF-1 in K28 cells.

    PubMed

    Park, Soo-Yun; Gomes, Cynthia; Oh, Sung-Dug; Soh, Jaemog

    2015-04-01

    Cadmium is a widely used heavy metal in industry and affects the male reproductive system of animals, including humans, as a result of occupational and environmental exposures. However, the molecular mechanism underlying its effect on steroidogenesis in gonads remains unclear. In this study, we demonstrated that exposure of K28 mouse testicular Leydig tumor cells to cadmium led to a significant increase in the mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis. It has been well documented that StAR gene transcription is regulated by multiple transcription factors, including cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused an increase in CREB phosphorylation but did not alter the CREB protein level in the nucleus. EMSA studies revealed that cadmium-induced phosphorylated CREB formed specific complexes with the proximal region of the StAR gene promoter. Furthermore, co-transfection with a CREB expression plasmid significantly increased cadmium-induced StAR promoter activity. However, the nuclear level and the affinity of SF-1 protein for the StAR proximal promoter were dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates StAR gene expression through phosphorylated CREB rather than through SF-1 in mouse testicular Leydig cells.

  17. Abundance of phosphorylated Apis mellifera CREB in the honeybee's mushroom body inner compact cells varies with age.

    PubMed

    Gehring, Katrin B; Heufelder, Karin; Kersting, Isabella; Eisenhardt, Dorothea

    2016-04-15

    Hymenopteran eusociality has been proposed to be associated with the activity of the transcription factor CREB (cAMP-response element binding protein). The honeybee (Apis mellifera) is a eusocial insect displaying a pronounced age-dependent division of labor. In honeybee brains, CREB-dependent genes are regulated in an age-dependent manner, indicating that there might be a role for neuronal honeybee CREB (Apis mellifera CREB, or AmCREB) in the bee's division of labor. In this study, we further explore this hypothesis by asking where in the honeybee brain AmCREB-dependent processes might take place and whether they vary with age in these brain regions. CREB is activated following phosphorylation at a conserved serine residue. An increase of phosphorylated CREB is therefore regarded as an indicator of CREB-dependent transcriptional activation. Thus, we here examine the localization of phosphorylated AmCREB (pAmCREB) in the brain and its age-dependent variability. We report prominent pAmCREB staining in a subpopulation of intrinsic neurons of the mushroom bodies. In these neurons, the inner compact cells (IC), pAmCREB is located in the nuclei, axons, and dendrites. In the central bee brain, the IC somata and their dendritic region, we observed an age-dependent increase of pAmCREB. Our results demonstrate the IC to be candidate neurons involved in age-dependent division of labor. We hypothesize that the IC display a high level of CREB-dependent transcription that might be related to neuronal and behavioral plasticity underlying a bee's foraging behavior.

  18. Activation of NR2A receptors induces ischemic tolerance through CREB signaling.

    PubMed

    Terasaki, Yasukazu; Sasaki, Tsutomu; Yagita, Yoshiki; Okazaki, Shuhei; Sugiyama, Yukio; Oyama, Naoki; Omura-Matsuoka, Emi; Sakoda, Saburo; Kitagawa, Kazuo

    2010-08-01

    Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

  19. Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

    PubMed Central

    Barbosa, Sónia; Carreira, Suzanne; Bailey, Daniel; Abaitua, Fernando; O'Hare, Peter

    2015-01-01

    CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1a E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV. PMID:26108621

  20. Long-term effects of early-life malnutrition and status epilepticus: assessment by spatial navigation and CREB(Serine-133) phosphorylation.

    PubMed

    Huang, Li-Tung; Lai, Ming-Chi; Wang, Chih-Lu; Wang, Chien-An; Yang, Chin-Hwa; Hsieh, Chih-Sung; Liou, Chia-Wei; Yang, San-Nan

    2003-11-12

    Malnutrition and/or seizure in the developing brain cause hippocampal damages. However, underlying mechanisms remain unclear. The malnutrition group (MN) subjected with malnutrition alone was culled to 20-22 rats per dam on postnatal day 1 (P1). The rats subjected to lithium-pilocarpine (Li/PC)-induced status epilepticus at P21 were grouped as the SE group. The rats subjected to malnutrition and subsequent status epilepticus were grouped as the MS group. Visual-spatial memory test using the Morris water maze task was performed at P80. Following behavioral tests, the hippocampus was evaluated for histological lesions and phosphorylated cAMP-responsive, element-binding protein at serine-133 (pCREB(Ser-133)), an important transcription factor underlying learning and memory in the mammalian brain. Here, the MN group exhibited decreased body weight at P21. There was no significant difference in the seizure duration and mortality between the SE and MS groups. In adulthood (P80), both the SE and MS groups showed the spatial learning deficit, hippocampal cell loss and decreased pCREB(Ser133) level within hippocampal CA1 region. Although the MN group demonstrated a decreased level of pCREB(Ser133), no distinguishable changes in the cognitive deficit and hippocampal neuronal loss were detected. Collectively, the present results suggest that early-life malnutrition led to a reduced phosphorylation of CREB(Ser133) in hippocampal CA1 in the absence of the long-term spatial learning deficit. This decreased phosphorylation of CREB(Ser133) could suggest that cascades of signal transduction responsible for the phosphorylation of CREB(Ser133) might be disturbed by early-life malnutrition. In addition, malnutrition caused no discernible synergistic effects on Li/PC-induced status epilepticus.

  1. CREB-Induced Inflammation Is Important for Malignant Mesothelioma Growth

    PubMed Central

    Westbom, Catherine M.; Shukla, Anurag; MacPherson, Maximilian B.; Yasewicz, Elizabeth C.; Miller, Jill M.; Beuschel, Stacie L.; Steele, Chad; Pass, Harvey I.; Vacek, Pamela M.; Shukla, Arti

    2015-01-01

    Malignant mesothelioma (MM) is an aggressive tumor with no treatment regimen. Previously we have demonstrated that cyclic AMP response element binding protein (CREB) is constitutively activated in MM tumor cells and tissues and plays an important role in MM pathogenesis. To understand the role of CREB in MM tumor growth, we generated CREB-inhibited MM cell lines and performed in vitro and in vivo experiments. In vitro experiments demonstrated that CREB inhibition results in significant attenuation of proliferation and drug resistance of MM cells. CREB-silenced MM cells were then injected into severe combined immunodeficiency mice, and tumor growth in s.c. and i.p. models of MM was followed. We observed significant inhibition in MM tumor growth in both s.c. and i.p. models and the presence of a chemotherapeutic drug, doxorubicin, further inhibited MM tumor growth in the i.p. model. Peritoneal lavage fluids from CREB-inhibited tumor-bearing mice showed a significantly reduced total cell number, differential cell counts, and pro-inflammatory cytokines and chemokines (IL-6, IL-8, regulated on activation normal T cell expressed and secreted, monocyte chemotactic protein-1, and vascular endothelial growth factor). In vitro studies showed that asbestos-induced inflammasome/inflammation activation in mesothelial cells was CREB dependent, further supporting the role of CREB in inflammation-induced MM pathogenesis. In conclusion, our data demonstrate the involvement of CREB in the regulation of MM pathogenesis by regulation of inflammation. PMID:25111229

  2. Curcumin confers neuroprotection against alcohol-induced hippocampal neurodegeneration via CREB-BDNF pathway in rats.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh; Fatima, Sulail; Hashemi, Hajar; Gholami, Mina

    2017-03-01

    Alcohol abuse causes severe damage to the brain neurons. Studies have reported the neuroprotective effects of curcumin against alcohol-induced neurodegeneration. However, the precise mechanism of action remains unclear. Seventy rats were equally divided into 7 groups (10 rats per group). Group 1 received normal saline (0.7ml/rat) and group 2 received alcohol (2g/kg/day) for 21days. Groups 3, 4, 5 and 6 concurrently received alcohol (2g/kg/day) and curcumin (10, 20, 40 and 60mg/kg, respectively) for 21days. Animals in group 7 self- administered alcohol for 21days. Group 8 treated with curcumin (60mg/kg, i.p.) alone for 21days. Open Field Test (OFT) was used to investigate motor activity in rats. Hippocampal oxidative, antioxidative and inflammatory factors were evaluated. Furthermore, brain cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and brain derived neurotrophic factor (BDNF) levels were studied at gene level by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, protein expression for BDNF, CREB, phosphorylated CREB (CREB-P), Bax and Bcl-2 was determined by western blotting. Voluntary and involuntary administration of alcohol altered motor activity in OFT, and curcumin treatment inhibited this alcohol-induced motor disturbance. Also, alcohol administration augmented lipid peroxidation, mitochondrial oxidized glutathione (GSSG), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and Bax levels in isolated hippocampal tissues. Furthermore, alcohol-induced significant reduction were observed in reduced form of glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and CREB, BDNF and Bcl-2 levels. Also curcumin alone did not change the behavior and biochemical and molecular parameters. Curcumin can act as a neuroprotective agent against neurodegenerative effects of alcohol abuse, probably via activation of CREB-BDNF signaling pathway

  3. CREB phosphorylation regulates striatal transcriptional responses in the self-administration model of methamphetamine addiction in the rat.

    PubMed

    Krasnova, Irina N; Chiflikyan, Margarit; Justinova, Zuzana; McCoy, Michael T; Ladenheim, Bruce; Jayanthi, Subramaniam; Quintero, Cynthia; Brannock, Christie; Barnes, Chanel; Adair, Jordan E; Lehrmann, Elin; Kobeissy, Firas H; Gold, Mark S; Becker, Kevin G; Goldberg, Steven R; Cadet, Jean Lud

    2013-10-01

    Neuroplastic changes in the dorsal striatum participate in the transition from casual to habitual drug use and might play a critical role in the development of methamphetamine (METH) addiction. We examined the influence of METH self-administration on gene and protein expression that may form substrates for METH-induced neuronal plasticity in the dorsal striatum. Male Sprague-Dawley rats self-administered METH (0.1mg/kg/injection, i.v.) or received yoked saline infusions during eight 15-h sessions and were euthanized 2h, 24h, or 1month after cessation of METH exposure. Changes in gene and protein expression were assessed using microarray analysis, RT-PCR and Western blots. Chromatin immunoprecipitation (ChIP) followed by PCR was used to examine epigenetic regulation of METH-induced transcription. METH self-administration caused increases in mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and the synaptic protein, synaptophysin (Syp) in the dorsal striatum. METH also caused changes in ΔFosB, BDNF and TrkB protein levels, with increases after 2 and 24h, but decreases after 1month of drug abstinence. Importantly, ChIP-PCR showed that METH self-administration caused enrichment of phosphorylated CREB (pCREB), but not of histone H3 trimethylated at lysine 4 (H3K4me3), on promoters of c-fos, fosb, Bdnf and Syp at 2h after cessation of drug intake. These findings show that METH-induced changes in gene expression are mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that mediate alterations in expression of genes and proteins serving as substrates for addiction-related synaptic plasticity.

  4. DPPC regulates COX-2 expression in monocytes via phosphorylation of CREB

    SciTech Connect

    Morris, R.H.K. Tonks, A.J.; Jones, K.P.; Ahluwalia, M.K.; Thomas, A.W.; Tonks, A.; Jackson, S.K.

    2008-05-23

    The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE{sub 2} (P < 0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-{kappa}B activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using L-{alpha}-phosphatidylcholine {beta}-arachidonoyl-{gamma}-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2.

  5. CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.

    EPA Science Inventory

    We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
    of C...

  6. CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.

    EPA Science Inventory

    We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
    of C...

  7. Changes in CREB activation in the prefrontal cortex and hippocampus blunt ethanol-induced behavioral sensitization in adolescent mice

    PubMed Central

    Soares-Simi, Sabrina L.; Pastrello, Daniel M.; Ferreira, Zulma S.; Yonamine, Mauricio; Marcourakis, Tania; Scavone, Cristoforo; Camarini, Rosana

    2013-01-01

    Drug dependence is a major health problem in adults and has been recognized as a significant problem in adolescents. We previously demonstrated that repeated treatment with a behaviorally sensitizing dose of ethanol in adult mice induced tolerance or no sensitization in adolescents and that repeated ethanol-treated adolescents expressed lower Fos and Egr-1 expression than adult mice in the prefrontal cortex (PFC). In the present work, we investigated the effects of acute and repeated ethanol administration on cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) DNA-binding activity using the electrophoretic mobility shift assay (EMSA) and the phosphorylated CREB (pCREB)/CREB ratio using immunoblotting in both the PFC and hippocampus in adolescent and adult mice. Adult mice exhibited typical locomotor sensitization after 15 days of daily treatment with 2.0 g/kg ethanol, whereas adolescent mice did not exhibit sensitization. Overall, adolescent mice displayed lower CREB binding activity in the PFC compared with adult mice, whereas opposite effects were observed in the hippocampus. The present results indicate that ethanol exposure induces significant and differential neuroadaptive changes in CREB DNA-binding activity in the PFC and hippocampus in adolescent mice compared with adult mice. These differential molecular changes may contribute to the blunted ethanol-induced behavioral sensitization observed in adolescent mice. PMID:24379765

  8. CREB Selectively Controls Learning-Induced Structural Remodeling of Neurons

    ERIC Educational Resources Information Center

    Middei, Silvia; Spalloni, Alida; Longone, Patrizia; Pittenger, Christopher; O'Mara, Shane M.; Marie, Helene; Ammassari-Teule, Martine

    2012-01-01

    The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons…

  9. CREB Selectively Controls Learning-Induced Structural Remodeling of Neurons

    ERIC Educational Resources Information Center

    Middei, Silvia; Spalloni, Alida; Longone, Patrizia; Pittenger, Christopher; O'Mara, Shane M.; Marie, Helene; Ammassari-Teule, Martine

    2012-01-01

    The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons…

  10. GSK-3 mediated phosphorylation couples ER-Golgi transport and nuclear stabilisation of the CREB-H transcription factor to mediate Apolipoprotein secretion.

    PubMed

    Barbosa, Sónia; Carreira, Suzanne; O'Hare, Peter

    2017-04-05

    CREB-H, an ER-anchored transcription factor plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases releasing the N-terminal product which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif, termed the P-motif located in the N-terminus between serines 73 to 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation promoting ER-retention while mutation of target serines or drug inhibition of GSK-3 activity, co-ordinately induces both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations which regulate stability by targeting the protein to the SCF(Fbw1a) E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilisation of the active form in the nucleus.

  11. Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

    PubMed

    Sakaguchi, H; Wada, K; Maekawa, M; Watsuji, T; Hagiwara, M

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning.

  12. CREB Phosphorylation Coincides with Transient Synapse Formation in the Rat Hippocampal Dentate Gyrus Following Avoidance Learning

    PubMed Central

    O'Connell, Cormac; Gallagher, Helen C.; O'Malley, Aoibheinn; Bourke, Mary; Regan, Ciaran M.

    2000-01-01

    Spine density change in the hippocampal dentate gyrus accompanies memory consolidation and coincides with the increased expression of ribosome-rich, hyperchromatic granule cells. Although this suggests increased protein synthesis to be required for synaptic growth in the 5 to 7 h post-training period, little temporal mapping of the associated molecular mechanisms has been done. Here, we demonstrate a similar frequency of hyperchromatic cells in naïve animals and in those sacrificed 6 h post-training, suggesting a transient repression of protein synthesis in the early post-training period. Immunoblot analysis of CREB phosphorylation in the dentate gyrus supported this view, with downregulation from basal levels observed at 2 to 3 h and at 12 h posttraining. Protein synthesis reactivation appears to be specific for de novo spine production as no change in spine frequency accompanies the immediate post-training period of depressed protein synthesis. These findings support the view that CREB-mediated gene transcription is a requirement for long-term memory consolidation and may be directly implicated in the process of synaptic growth. PMID:11486487

  13. A high-fat diet or galanin in the PVN decreases phosphorylation of CREB in the nucleus accumbens

    PubMed Central

    Bocarsly, Miriam E.; Avena, Nicole M.

    2013-01-01

    A high-fat diet (HFD) can increase hypothalamic galanin (GAL). GAL has recently been shown to inhibit opiate reward, which in turn, decreases cAMP response element-binding protein (CREB) in the nucleus accumbens (NAc). We hypothesized that injection of GAL into the PVN, or consumption of a HFD, would be associated with a decrease in NAc CREB. In Exp. 1, GAL in the paraventricular nucleus (PVN) of naïve rats decreased phosphorylated-CREB (pCREB) in the NAc compared to saline injected controls. In Exp. 2, rats fed ad libitum HFD for 4 wks had reduced NAc pCREB levels compared to rats with sporadic tastes of the HFD. Body weight, serum triglyceride and leptin levels were also raised in the chronic HFD-fed rats. These data suggest that PVN GAL or chronic intake of a HFD can decrease NAc pCREB. The implications of these findings may help to explain the lack of opiate-like withdrawal that has been reported in response to overeating a high fat diet, thereby providing a potential mechanism underlying behavioral differences seen with addiction-like overconsumption of different types of palatable foods. PMID:23747305

  14. Scopoletin from Cirsium setidens Increases Melanin Synthesis via CREB Phosphorylation in B16F10 Cells.

    PubMed

    Ahn, Mi-Ja; Hur, Sun-Jung; Kim, Eun-Hyun; Lee, Seung Hoon; Shin, Jun Seob; Kim, Myo-Kyoung; Uchizono, James A; Whang, Wan-Kyunn; Kim, Dong-Seok

    2014-08-01

    In this study, we isolated scopoletin from Cirsium setidens Nakai (Compositae) and tested its effects on melanogenesis. Scopoletin was not toxic to cells at concentrations less than 50 µM and increased melanin synthesis in a dose-dependent manner. As melanin synthesis increased, scopoletin stimulated the total tyrosinase activity, the rate-limiting enzyme of melanogenesis. In a cell-free system, however, scopoletin did not increase tyrosinase activity, indicating that scopoletin is not a direct activator of tyrosinase. Furthermore, Western blot analysis showed that scopoletin stimulated the production of microphthalmia-associated transcription factor (MITF) and tyrosinase expression via cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Based on these results, preclinical and clinical studies are needed to assess the use of scopoletin for the treatment of vitiligo.

  15. Scopoletin from Cirsium setidens Increases Melanin Synthesis via CREB Phosphorylation in B16F10 Cells

    PubMed Central

    Ahn, Mi-Ja; Hur, Sun-Jung; Kim, Eun-Hyun; Lee, Seung Hoon; Shin, Jun Seob; Kim, Myo-Kyoung; Uchizono, James A.; Whang, Wan-Kyunn

    2014-01-01

    In this study, we isolated scopoletin from Cirsium setidens Nakai (Compositae) and tested its effects on melanogenesis. Scopoletin was not toxic to cells at concentrations less than 50 µM and increased melanin synthesis in a dose-dependent manner. As melanin synthesis increased, scopoletin stimulated the total tyrosinase activity, the rate-limiting enzyme of melanogenesis. In a cell-free system, however, scopoletin did not increase tyrosinase activity, indicating that scopoletin is not a direct activator of tyrosinase. Furthermore, Western blot analysis showed that scopoletin stimulated the production of microphthalmia-associated transcription factor (MITF) and tyrosinase expression via cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Based on these results, preclinical and clinical studies are needed to assess the use of scopoletin for the treatment of vitiligo. PMID:25177162

  16. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    SciTech Connect

    Hwang, Tsong-Long; Tang, Ming-Chi; Kuo, Liang-Mou; Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching

    2012-04-15

    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  17. Dorsal hippocampal NMDA receptor blockade impairs extinction of naloxone-precipitated conditioned place aversion in acute morphine-treated rats by suppressing ERK and CREB phosphorylation in the basolateral amygdala

    PubMed Central

    Wang, Wei-Sheng; Chen, Zhong-Guo; Liu, Wen-Tao; Chi, Zhi-Qiang; He, Ling; Liu, Jing-Gen

    2015-01-01

    BACKGROUND AND PURPOSE Substantial evidence shows that negative reinforcement resulting from the aversive affective consequences of opiate withdrawal may play a crucial role in drug relapse. Understanding the mechanisms underlying the loss (extinction) of conditioned aversion of drug withdrawal could facilitate the treatment of drug addiction. EXPERIMENTAL APPROACH Naloxone-induced conditioned place aversion (CPA) of Sprague-Dawley rats was used to measure conditioned aversion. An NMDA receptor antagonist and MAPK kinase inhibitor were applied through intracranial injections. The phosphorylation of ERK and cAMP response element-binding protein (CREB) was detected using Western blot. KEY RESULTS The extinction of CPA behaviour increased the phosphorylation of ERK and CREB in the dorsal hippocampus (DH) and basolateral amygdala (BLA), but not in the central amygdala (CeA). Intra-DH injection of AP5 or intra-BLA injection of AP-5 or U0126 before extinction training significantly attenuated ERK and CREB phosphorylation in the BLA and impaired the extinction of CPA behaviour. Although intra-DH injections of AP-5 attenuated extinction training-induced activation of the ERK-CREB pathway in the BLA, intra-BLA injection of AP5 had no effect on extinction training-induced activation of the ERK-CREB pathway in the DH. CONCLUSIONS AND IMPLICATIONS These results suggest that activation of ERK and CREB in the BLA and DH is involved in the extinction of CPA behaviour and that the DH, via a direct or indirect pathway, modulates the activity of ERK and CREB in the BLA through activation of NMDA receptors after extinction training. Understanding the mechanisms underlying the extinction of conditioned aversion could facilitate the treatment of drug addiction. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24597568

  18. Exercise Prevents Memory Impairment Induced by Arsenic Exposure in Mice: Implication of Hippocampal BDNF and CREB

    PubMed Central

    Yu, Zi-Jiang; Yu, Yan; Xiao, Chao-Lun; Kang, Chao-Sheng; Ge, Guo; Linghu, Yan; Zhu, Jun-De; Li, Yu-Mei; Li, Qiang-Ming; Luo, Shi-Peng; Yang, Dang; Li, Lin; Zhang, Wen-Yan; Tian, Guang

    2015-01-01

    High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus. PMID:26368803

  19. Exercise Prevents Memory Impairment Induced by Arsenic Exposure in Mice: Implication of Hippocampal BDNF and CREB.

    PubMed

    Sun, Bao-Fei; Wang, Qing-Qing; Yu, Zi-Jiang; Yu, Yan; Xiao, Chao-Lun; Kang, Chao-Sheng; Ge, Guo; Linghu, Yan; Zhu, Jun-De; Li, Yu-Mei; Li, Qiang-Ming; Luo, Shi-Peng; Yang, Dang; Li, Lin; Zhang, Wen-Yan; Tian, Guang

    2015-01-01

    High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus.

  20. Notch-inducible hyperphosphorylated CREB and its ultradian oscillation in long-term memory formation.

    PubMed

    Zhang, Jiabin; Little, Christopher J; Tremmel, Daniel M; Yin, Jerry C P; Wesley, Cedric S

    2013-07-31

    Notch is a cell surface receptor that is known to regulate developmental processes by establishing physical contact between neighboring cells. Many recent studies show that it also plays an important role in the formation of long-term memory (LTM) in adults, implying that memory formation requires regulation at the level of cell-cell contacts among brain cells. Neither the target of Notch activity in LTM formation nor the underlying mechanism of regulation is known. We report here results of our studies in adult Drosophila melanogaster showing that Notch regulates dCrebB-17A, the CREB protein. CREB is a transcriptional factor that is pivotal for intrinsic and synaptic plasticity involved in LTM formation. Notch in conjunction with PKC activity upregulates the level of a hyperphosphorylated form of CREB (hyper-PO4 CREB) and triggers its ultradian oscillation, both of which are linked to LTM formation. One of the sites that is phosphorylated in hyper-PO4 CREB is serine 231, which is the functional equivalent of mammalian CREB serine 133, the phosphorylation of which is an important regulator of CREB functions. Our data suggest the model that Notch and PKC activities generate a cyclical accumulation of cytoplasmic hyper-PO4 CREB that is a precursor for generating the nuclear CREB isoforms. Cyclical accumulation of CREB might be important for repetitive aspects of LTM formation, such as memory consolidation. Because Notch, PKC, and CREB have been implicated in many neurodegenerative diseases (e.g., Alzheimer's disease), our data might also shed some light on memory loss and dementia.

  1. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

    PubMed Central

    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

    2013-01-01

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

  2. Running exercise-induced up-regulation of hippocampal brain-derived neurotrophic factor is CREB-dependent

    PubMed Central

    Chen, Michael J.; Russo-Neustadt, Amelia A.

    2009-01-01

    The past decade has witnessed burgeoning evidence that antidepressant medications and physical exercise increase the expression of hippocampal brain-derived neurotrophic factor (BDNF). This phenomenon has gained widespread appeal because BDNF is one of the first macromolecules observed to play a central role not only in the treatment of mood disorders, but also in neuronal survival-, growth-, and plasticity-related signaling cascades. Thus, it has become critical to understand how BDNF synthesis is regulated. Much evidence exists that changes in BDNF expression result from the activation/phosphorylation of the transcription factor, cAMP-response-element binding protein (CREB) following the administration of antidepressant medications. Utilizing a mouse model genetically engineered with an inducible CREB repressor, our current study provides evidence that increases in BDNF expression and cellular survival signaling resulting from physical exercise are also dependent upon activation of this central transcription factor. The transcription and expression of hippocampal BDNF, as well as the activation of Akt, a key survival signaling molecule, were measured following acute exercise, and also following short-term treatment with the norepinephrine re-uptake inhibitor, reboxetine. We found that both interventions led to a marked increase in hippocampal BDNF mRNA, BDNF protein and Akt phosphorylation (as well as CREB phosphorylation) in wild-type mice. As expected, activation of the CREB repressor in mutant mice sharply decreased CREB phosphorylation. In addition, all measures noted above remained at baseline levels when mutant mice exercised or received reboxetine. Increases in BDNF and phospho-Akt were also prevented when mutant mice received a combination of exercise and antidepressant treatment. The results are discussed in the context of what is currently known about BDNF signaling. PMID:19294650

  3. Running exercise-induced up-regulation of hippocampal brain-derived neurotrophic factor is CREB-dependent.

    PubMed

    Chen, Michael J; Russo-Neustadt, Amelia A

    2009-10-01

    The past decade has witnessed burgeoning evidence that antidepressant medications and physical exercise increase the expression of hippocampal brain-derived neurotrophic factor (BDNF). This phenomenon has gained widespread appeal, because BDNF is one of the first macromolecules observed to play a central role not only in the treatment of mood disorders, but also in neuronal survival-, growth-, and plasticity-related signaling cascades. Thus, it has become critical to understand how BDNF synthesis is regulated. Much evidence exists that changes in BDNF expression result from the activation/phosphorylation of the transcription factor, cAMP-response-element binding protein (CREB) following the administration of antidepressant medications. Utilizing a mouse model genetically engineered with an inducible CREB repressor, our current study provides evidence that increases in BDNF expression and cellular survival signaling resulting from physical exercise are also dependent upon activation of this central transcription factor. The transcription and expression of hippocampal BDNF, as well as the activation of Akt, a key survival signaling molecule, were measured following acute exercise, and also following short-term treatment with the norepinephrine reuptake inhibitor, reboxetine. We found that both interventions led to a marked increase in hippocampal BDNF mRNA, BDNF protein, and Akt phosphorylation (as well as CREB phosphorylation) in wild-type mice. As expected, activation of the CREB repressor in mutant mice sharply decreased CREB phosphorylation. In addition, all measures noted above remained at baseline levels when mutant mice exercised or received reboxetine. Increases in BDNF and phospho-Akt were also prevented when mutant mice received a combination of exercise and antidepressant treatment. The results are discussed in the context of what is currently known about BDNF signaling.

  4. Epinephrine and glucose modulate training-related CREB phosphorylation in old rats: relationships to age-related memory impairments.

    PubMed

    Morris, Ken A; Gold, Paul E

    2013-02-01

    Epinephrine enhances memory in young adult rats, in part, by increasing blood glucose levels needed to modulate memory. In old rats, epinephrine is deficient at raising blood glucose levels and thus is only moderately effective at enhancing memory. In contrast, systemic glucose injections improve memory in old rats, with resulting memory performance equal to that of young rats. The diminished response of glucose to training in old rats may blunt downstream neurochemical and molecular mechanisms needed to upregulate memory processes. In the first experiment, young adult and old rats were trained on an inhibitory avoidance task with immediate post-training injections of aCSF or glucose into the dorsal hippocampus. Old rats had significant memory impairments compared to young rats 7 days after training. Intrahippocampal injections of glucose reversed age-related deficits, improving memory scores in old rats to values seen in young rats. A second experiment examined age-related changes in activation of the transcription factor CREB, which is widely implicated in memory formation and may act downstream of hormonal and metabolic signals. Activation was assessed in response to training with systemic injections of epinephrine and glucose at doses known to enhance memory. Young adult and old rats were trained on inhibitory avoidance with immediate post-training systemic injections of saline, epinephrine, or glucose. After training, old rats had significant impairments in CREB phosphorylation in area CA1 and the dentate gyrus region of the hippocampus, and in the basolateral and lateral amygdala. Epinephrine and glucose attenuated age-related deficits in CREB phosphorylation, but were more effective in the amygdala and hippocampus, respectively. Together, these results support the view that age-related changes in blood glucose responses to epinephrine contribute to memory impairments, which may be related to alterations in regional patterns of CREB phosphorylation.

  5. EPINEPHRINE AND GLUCOSE MODULATE TRAINING-RELATED CREB PHOSPHORYLATION IN OLD RATS: RELATIONSHIPS TO AGE-RELATED MEMORY IMPAIRMENTS

    PubMed Central

    Morris, Ken A.; Gold, Paul E.

    2012-01-01

    Epinephrine enhances memory in young adult rats, in part, by increasing blood glucose levels needed to modulate memory. In old rats, epinephrine is deficient at raising blood glucose levels and thus is only moderately effective at enhancing memory. In contrast, systemic glucose injections improve memory in old rats, with resulting memory performance equal to that of young rats. The diminished response of glucose to training in old rats may blunt downstream neurochemical and molecular mechanisms needed to upregulate memory processes. In the first experiment, young adult and old rats were trained on an inhibitory avoidance task with immediate post-training injections of aCSF or glucose into the dorsal hippocampus. Old rats had significant memory impairments compared to young rats 7 days after training. Intrahippocampal injections of glucose reversed age-related deficits, improving memory scores in old rats to values seen in young rats. A second experiment examined age-related changes in activation of the transcription factor CREB, which is widely implicated in memory formation and may act downstream of hormonal and metabolic signals. Activation was assessed in response to training with systemic injections of epinephrine and glucose at doses known to enhance memory. Young adult and old rats were trained on inhibitory avoidance with immediate post-training systemic injections of saline, epinephrine, or glucose. After training, old rats had significant impairments in CREB phosphorylation in area CA1 and the dentate gyrus region of the hippocampus, and in the basolateral and lateral amygdala. Epinephrine and glucose attenuated age-related deficits in CREB phosphorylation, but were more effective in the amygdala and hippocampus, respectively. Together, these results support the view that age-related changes in blood glucose responses to epinephrine contribute to memory impairments, which may be related to alterations in regional patterns of CREB phosphorylation. PMID

  6. Increased phosphorylation of Ser473-Akt, Ser9-GSK-3beta and Ser133-CREB in the rat frontal cortex after MK-801 intraperitoneal injection.

    PubMed

    Ahn, Yong Min; Seo, Myoung Suk; Kim, Se Hyun; Kim, Yeni; Yoon, Se Chang; Juhnn, Yong-Sung; Kim, Yong Sik

    2005-12-01

    GSK-3beta is regarded as playing an important part in the pathogenesis of schizophrenia and the action of psychotomimetic agents. We observed phosphorylation of molecules associated with the GSK-3beta signalling pathway in the rat brain after MK-801 injection, which induces a schizophrenia-like state in humans. Ser9-GSK-3beta phosphorylation was increased after injection of 1 mg/kg MK-801 in the rat frontal cortex but not in the hippocampus or cerebellum. This increase peaked at 30 min and was maintained until 90 min after injection. The phosphorylation showed a dose-dependent increase up to 1 mg/kg MK-801, followed by a decrease at higher dosage. Furthermore, phosphorylation of Ser473-Akt and Ser133-CREB showed similar temporal, dose-dependent and regionally specific patterns with those of Ser9-GSK-3beta. However, phosphorylation of Dvl and Ser33-beta-catenin was not affected by MK-801. These results suggest that GSK-3beta phosphorylation by MK-801 may be associated with the Akt-GSK-3beta pathway rather than with the Wnt-Dvl-GSK3beta pathway.

  7. PERK mediates eIF2α phosphorylation responsible for BACE1 elevation, CREB dysfunction and neurodegeneration in a mouse model of Alzheimer's disease.

    PubMed

    Devi, Latha; Ohno, Masuo

    2014-10-01

    Emerging evidence suggests that aberrant phosphorylation of eukaryotic initiation factor-2α (eIF2α) may induce synaptic failure and neurodegeneration through persistent translational inhibition of global protein synthesis. However, elevated phospho-eIF2α also paradoxically causes translational activation of a subset of messenger RNAs such as the β-secretase enzyme, β-site APP-cleaving enzyme 1 (BACE1) and cAMP response element binding protein (CREB) repressor, activating transcription factor 4 (ATF4). Therefore, we tested whether genetic reduction of the eIF2α kinase PERK may prevent these deleterious events and mitigate Alzheimer's disease (AD)-like neuropathology and cognitive impairments in the 5XFAD mouse model. PERK haploinsufficiency blocked overactivation of the PERK-eIF2α pathway, as evidenced by significant reductions in phosphorylation of PERK and eIF2α, in 5XFAD mice. PERK haploinsufficiency was sufficient to rescue memory deficits and cholinergic neurodegeneration in this AD model. Notably, PERK haploinsufficiency also prevented BACE1 elevations, resulting in reduced levels of amyloid-β peptides and plaque burden in 5XFAD mice. Moreover, CREB dysfunction was restored in PERK(+/-)·5XFAD mice concomitant with reversal of ATF4 upregulation. Together, these findings suggest that PERK may be a disease-modifying therapeutic target to prevent multiple memory-disrupting mechanisms associated with AD.

  8. Trigeminal Neuralgia Induced by Cobra Venom Leads to Cognitive Deficits Associated with Downregulation of CREB/BDNF Pathway.

    PubMed

    Zhang, Li; Ding, Xinli; Wu, Zhe; Qian, Xiaoyan; An, Jianxiong; Tian, Ming

    2017-02-01

    Chronic pain often results in cognitive impairment. Our previous study showed that trigeminal neuralgia induced by cobra venom leads to spatial learning and memory deficits, although the underlying mechanism remains unclear. However, recent evidence indicates that the c-AMP-responsive element binding protein (CREB)/brain derived neurotrophic factor (BDNF) pathway plays a critical role in various etiologies of cognitive deficits. Our aim was to explore the CREB/BDNF pathway to determine the molecular mechanisms involved in the pathogenesis of cognitive impairment caused by cobra venom-induced trigeminal neuralgia. A randomized, controlled animal study. Department of Anesthesiology, Beijing Friendship Hospital, Capital Medical University. Fifty male Sprague-Dawley rats were randomly divided into 3 groups: cobra venom group, sham group, and control group. Cobra venom or saline was injected into the sheath of the infraorbital nerve (ION), respectively. Video recordings and mechanical thresholds were used to analyze changes in behavioral activity 3 days before surgery and 4, 7, 14, 21, 28, and 56 days after surgery. Morris water maze tests were conducted at 4- and 8-week time points after surgery to evaluate spatial learning and memory. We also investigated expression changes of phosphorylated CREB (p-CREB) and BDNF in the hippocampus and prefrontal cortex (PFC) using western blotting and immunohistochemistry. Cobra venom-treated rats exhibited significant changes in face grooming, as well as exploratory and resting behaviors, compared with the control group and sham group (both P < 0.001). Rats in the cobra venom group exhibited slightly impaired acquisition (P < 0.05) without memory deficits (P > 0.05) in the first water maze protocol. In the second water maze test, rats in the cobra venom group exhibited spatial learning and memory deficits, with fewer platform site crossings during the probe trial (P < 0.05). Moreover, results showed decreased p-CREB and BDNF

  9. Peptide IDR-1002 Inhibits NF-κB Nuclear Translocation by Inhibition of IκBα Degradation and Activates p38/ERK1/2-MSK1-Dependent CREB Phosphorylation in Macrophages Stimulated with Lipopolysaccharide.

    PubMed

    Huante-Mendoza, Alejandro; Silva-García, Octavio; Oviedo-Boyso, Javier; Hancock, Robert E W; Baizabal-Aguirre, Víctor M

    2016-01-01

    lead to expression of IL-4, IL-10, and IL-13, even though it induced an increase in phospho-CREB relative abundance. In conclusion, our results indicated that IDR-1002 has a dual effect. On one hand, it inhibited NF-κB nuclear translocation through a mechanism that involved inhibition of IκBα phosphorylation, and on the other, it activated a protein kinase signaling cascade that phosphorylated CREB to selectively influence cytokine gene expression. Based on these results, we think IDR-1002 could be a potential good biopharmaceutical candidate to control inflammation.

  10. Peptide IDR-1002 Inhibits NF-κB Nuclear Translocation by Inhibition of IκBα Degradation and Activates p38/ERK1/2–MSK1-Dependent CREB Phosphorylation in Macrophages Stimulated with Lipopolysaccharide

    PubMed Central

    Huante-Mendoza, Alejandro; Silva-García, Octavio; Oviedo-Boyso, Javier; Hancock, Robert E. W.; Baizabal-Aguirre, Víctor M.

    2016-01-01

    not lead to expression of IL-4, IL-10, and IL-13, even though it induced an increase in phospho-CREB relative abundance. In conclusion, our results indicated that IDR-1002 has a dual effect. On one hand, it inhibited NF-κB nuclear translocation through a mechanism that involved inhibition of IκBα phosphorylation, and on the other, it activated a protein kinase signaling cascade that phosphorylated CREB to selectively influence cytokine gene expression. Based on these results, we think IDR-1002 could be a potential good biopharmaceutical candidate to control inflammation. PMID:27933067

  11. Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

    PubMed Central

    Wilson, B E; Mochon, E; Boxer, L M

    1996-01-01

    Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during

  12. Differential effects of chronic amphetamine and baclofen administration on cAMP levels and phosphorylation of CREB in distinct brain regions of wild type and monoamine oxidase B-deficient mice.

    PubMed

    Yin, Hsiang-Shu; Chen, Kevin; Kalpana, Sriram; Shih, Jean C

    2006-12-15

    Roles of GABA(B) transmission were explored in the action of amphetamine (Amph) on the brain. Adult male wild type (WT) and monoamine oxidase B-knocked out (MAOBKO) mice received i.p. injections of saline, d-Amph (5 mg/kg), plus baclofen (GABA(B) receptor agonist, 10 mg/kg), or baclofen and Amph, twice daily for 3 days and single treatments on day 4, followed by immuno-cyclic-AMP (cAMP) and immunoblotting assays on the brain tissue. The WT mice responded with higher levels of behavioral responses than the KO to the daily Amph injection; however, baclofen blocked the Amph-induced behavioral hyperactivity of both WT and KO mice. After the last treatment, levels of cAMP and phosphorylated (p) cyclic-AMP response element binding protein (CREB) were up-regulated in the striatum and somatosensory cortex of Amph-treated WT mice, while similar to the saline-controls in the baclofen+Amph-treated group, indicating the blockade by baclofen to Amph. Baclofen similarly suppressed the Amph-induced increases in pCREB levels of WT hippocampus and amygdala, and decreases of olfactory bulb and thalamus. For MAOBKO mice, baclofen hindered the Amph-generated increases in motor cortical cAMP and pCREB, and amygdaloid pCREB, and the decrease in olfactory bulb pCREB, whereas did not affect the Amph-raised hippocampal pCREB. Furthermore, the levels of CREB were variably modified in distinct regions by the drug exposures. The data reveal that the GABA(B)-mediated intracellular signaling differentially participates in mechanisms underlying Amph perturbation to various regions, and may thereby contribute explanations to the behavioral consequences. Moreover, MAOB is region-dependently involved in responses of the brain to Amph and baclofen, supporting interactions between GABA and monoamines.

  13. Association of MMP7 -181A→G Promoter Polymorphism with Gastric Cancer Risk: INFLUENCE OF NICOTINE IN DIFFERENTIAL ALLELE-SPECIFIC TRANSCRIPTION VIA INCREASED PHOSPHORYLATION OF cAMP-RESPONSE ELEMENT-BINDING PROTEIN (CREB).

    PubMed

    Kesh, Kousik; Subramanian, Lakshmi; Ghosh, Nillu; Gupta, Vinayak; Gupta, Arnab; Bhattacharya, Samir; Mahapatra, Nitish R; Swarnakar, Snehasikta

    2015-06-05

    Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.

  14. Leptin Induces Hippocampal Synaptogenesis via CREB-Regulated MicroRNA-132 Suppression of p250GAP

    PubMed Central

    Dhar, Matasha; Zhu, Mingyan; Impey, Soren; Lambert, Talley J.; Bland, Tyler; Karatsoreos, Ilia N.; Nakazawa, Takanobu

    2014-01-01

    Leptin acts in the hippocampus to enhance cognition and reduce depression and anxiety. Cognitive and emotional disorders are associated with abnormal hippocampal dendritic spine formation and synaptogenesis. Although leptin has been shown to induce synaptogenesis in the hypothalamus, its effects on hippocampal synaptogenesis and the mechanism(s) involved are not well understood. Here we show that leptin receptors (LepRs) are critical for hippocampal dendritic spine formation in vivo because db/db mice lacking the long form of the leptin receptor (LepRb) have reduced spine density on CA1 and CA3 neurons. Leptin promotes the formation of mature spines and functional glutamate synapses on hippocampal pyramidal neurons in both dissociated and slice cultures. These effects are blocked by short hairpin RNAs specifically targeting the LepRb and are absent in cultures from db/db mice. Activation of the LepR leads to cAMP response element–binding protein (CREB) phosphorylation and initiation of CREB-dependent transcription via the MAPK kinase/Erk pathway. Furthermore, both Mek/Erk and CREB activation are required for leptin-induced synaptogenesis. Leptin also increases expression of microRNA-132 (miR132), a well-known CREB target, which is also required for leptin-induced synaptogenesis. Last, leptin suppresses the expression of p250GAP, a miR132 target, and this suppression is obligatory for leptin's effects as is the downstream target of p250GAP, Rac1. LepRs appear to be critical in vivo as db/db mice have lowered hippocampal miR132 levels and elevated p250GAP expression. In conclusion, we identify a novel signaling pathway by which leptin increases synaptogenesis through inducing CREB transcription and increasing microRNA-mediated suppression of p250GAP activity, thus removing a known inhibitor of Rac1-stimulated synaptogenesis. PMID:24877561

  15. Heat induces interleukin-6 in skeletal muscle cells via TRPV1/PKC/CREB pathways.

    PubMed

    Obi, Syotaro; Nakajima, Toshiaki; Hasegawa, Takaaki; Kikuchi, Hironobu; Oguri, Gaku; Takahashi, Masao; Nakamura, Fumitaka; Yamasoba, Tatsuya; Sakuma, Masashi; Toyoda, Shigeru; Tei, Chuwa; Inoue, Teruo

    2017-03-01

    Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37-42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6

  16. Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

    PubMed Central

    Bottazzi, Maria Elena; Buzzai, Monica; Zhu, Xiaoyun; Desdouets, Chantal; Bréchot, Christian; Assoian, Richard K.

    2001-01-01

    Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G1-phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation. PMID:11604497

  17. γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression

    PubMed Central

    Ma, Huan; Groth, Rachel D.; Cohen, Samuel M.; Emery, John F.; Li, Bo-Xing; Hoedt, Esthelle; Zhang, Guo-An; Neubert, Thomas A.; Tsien, Richard W.

    2014-01-01

    SUMMARY Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events but how information is relayed onward to the nucleus remains unclear. Here we report a novel mechanism that mediates long-distance communication within cells: a shuttle that transports Ca2+/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, that its phosphorylation at Thr287 by βCaMKII protects the Ca2+/CaM signal, and that CaN triggers its nuclear translocation. Both βCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, while γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca2+/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves longstanding puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII and CaN in multiple neuropsychiatric disorders. PMID:25303525

  18. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    PubMed

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors.

  19. Peroxisome proliferator-activated receptor-γ (PPARγ) agonist is neuroprotective and stimulates PGC-1α expression and CREB phosphorylation in human dopaminergic neurons.

    PubMed

    Mäkelä, Johanna; Tselykh, Timofey V; Kukkonen, Jyrki P; Eriksson, Ove; Korhonen, Laura T; Lindholm, Dan

    2016-03-01

    Mitochondrial dysfunction has been linked to several neurodegenerative diseases such as Parkinson's disease (PD). Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master gene for mitochondrial biogenesis and has been shown to be neuroprotective in models of PD. In this work we have studied the mechanisms by which peroxisome proliferator-activated receptor-γ (PPARγ) selective agonist N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine hydrate (GW1929) acts on human dopaminergic neurons in culture. Data showed that GW1929 increased the viability of human dopaminergic neurons and protected them against oxidative stress induced by H2O2 and the mitochondrial toxin Rotenone. The enhanced resilience of the neurons was attributed to increased levels of mitochondrial antioxidants and of PGC-1α. GW1929 treatment further increased cell respiration, mitochondrial biogenesis and sirtuin-1 (SIRT1) expression in the human dopaminergic neurons. Phosphorylation of cAMP responsive element-binding protein (CREB) was also robustly increased in GW1929-treated cells. Together these results show that the PPARγ agonist GW1929 influences CREB signaling and PGC-1α activities in the human dopaminergic neurons contributing to an increased cell viability. This supports the view that drugs acting on the PPARγ-PGC-1α signaling in neurons may have beneficial effects in PD and possible also in other brain disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Involvement of the cAMP Response Element Binding Protein, CREB, and Cyclin D1 in LPA-Induced Proliferation of P19 Embryonic Carcinoma Cells

    PubMed Central

    Kim, Min-Jung; Sun, Yuanjie; Yang, Haijie; Kim, Nam-Ho; Jeon, Sung Ho; Huh, Sung-Oh

    2012-01-01

    Lysophosphatidic acid (LPA) is a lipid growth factor that induces proliferation of fibroblasts by activating the cAMP response element binding protein (CREB). Here, we further investigated whether LPA induces proliferation of P19 cells, a line of pluripotent embryonic carcinoma cells. 5′-Bromo-2-deoxyuridine incorporation and cell viability as-says showed that LPA stimulated proliferation of P19 cells. Immunoblot experiments with P19 cells revealed that the mitogen activated protein kinases, including p-ERK, p38, pAKT, glycogen synthase kinase 3β, and CREB were phosphorylated by treatment with 10 μM LPA. LPA-induced phosphorylation of CREB was efficiently blocked by U0126 and H89, inhibitors of the MAP kinases ERK1/2 and mitogen- and stress-activated protein kinase 1, respectively. Involvement of cyclin D1 in LPA-induced P19 cell proliferation was verified by immunoblot analysis in combination with pharmacological inhibitor treatment. Furthermore, LPA up-regulated CRE-harboring cyclin D1 promoter activity, suggesting that CREB and cyclin D1 play significant roles in LPA-induced proliferation of P19 embryonic carcinoma cells. PMID:22847216

  1. Reduced CREB phosphorylation after chronic lithium treatment is associated with down-regulation of CaM kinase IV in rat hippocampus.

    PubMed

    Tardito, Daniela; Tiraboschi, Ettore; Kasahara, Jiro; Racagni, Giorgio; Popoli, Maurizio

    2007-08-01

    Lithium is widely used in the treatment of bipolar disorder, although its mechanism of action is not fully clear. This study was undertaken to assess the effects of prolonged lithium administration on cAMP responsive element-binding protein (CREB) phosphorylation and CaM kinase IV (CaMKIV), one of the main kinases phosphorylating CREB in neurons following synaptic activation. CREB total protein expression and phosphorylation (Ser133), as well as CaMKIV enzymatic activity, phosphorylation of Thr196 (the activator residue) and kinase expression level were assessed in total homogenates and nuclei from the hippocampus and prefrontal/frontal cortex following 5 wk lithium treatment. Whereas no significant effects were found in prefrontal/frontal cortex, lithium administration reduced CREB phosphorylation and at the same time down-regulated CaMKIV (enzymatic activity, phospho-Thr196 and protein expression level) in cell nuclei from the hippocampus. These data suggest for the first time the involvement of CaMKIV in the mechanism of action of lithium.

  2. CREB regulates TNF-α-induced GM-CSF secretion via p38 MAPK in human lung fibroblasts.

    PubMed

    Koga, Yasuhiko; Hisada, Takeshi; Ishizuka, Tamotsu; Utsugi, Mitsuyoshi; Ono, Akihiro; Yatomi, Masakiyo; Kamide, Yosuke; Aoki-Saito, Haruka; Tsurumaki, Hiroaki; Dobashi, Kunio; Yamada, Masanobu

    2016-10-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that mediates eosinophilic differentiation, migration and survival, causing respiratory tract inflammation. GM-CSF is also known to be secreted from respiratory tract structural cells. However, the mechanisms of GM-CSF secretion have not been well established. Human fetal lung fibroblasts and human primary asthmatic lung fibroblasts were used for the study of tumor necrosis factor alpha (TNF-α)-induced GM-CSF secretion. GM-CSF secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction, respectively. Knockdown of cAMP response element-binding protein (CREB) in fibroblasts was carried out by using specific small interfering RNAs of CREB. Among respiratory tract structural cells, pulmonary fibroblasts exhibited increased GM-CSF secretion and mRNA expression after stimulation with TNF-α in a concentration-dependent manner. Moreover, a p38 mitogen-activated protein kinase (MAPK) inhibitor controlled TNF-α-induced GM-CSF secretion, and roflumilast and rolipram, inhibitors of phosphodiesterase-4, suppressed TNF-α-induced GM-CSF secretion. Consistent with this, forskolin also completely blocked GM-CSF secretion, and similar results were observed in response to cAMP treatment, suggesting that cAMP signaling suppressed TNF-α-induced GM-CSF secretion in human lung fibroblasts. Furthermore, CREB was phosphorylated through p38 MAPK but not cAMP signaling after TNF-α stimulation, and GM-CSF secretion was inhibited by CREB knockdown. Finally, these effects were also demonstrated in human primary lung fibroblasts in a patient with asthma. CREB signaled independent of cAMP signaling and was phosphorylated by p38 MAPK following TNF-α stimulation, playing a critical role in GM-CSF secretion in human lung fibroblasts. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All

  3. Prevention of vascular smooth muscle cell proliferation and injury-induced neointimal hyperplasia by CREB-mediated p21 induction: An insight from a plant polyphenol.

    PubMed

    Sun, Lan; Zhao, Rui; Zhang, Li; Zhang, Weiku; He, GuoRong; Yang, Shengqian; Song, Junke; Du, Guanhua

    2016-03-01

    Cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element (CRE)-binding protein (CREB) signaling cascade negatively regulates platelet-derived growth factor BB (PDGF-BB)-induced smooth muscle cell (SMC) proliferation, which is a critical event in the initiation and development of restenosis and atherosclerotic lesions. Salvianolic acid A (SAA) is one of the most abundant polyphenols extracted from salvia. The aim of this study is to investigate whether SAA exerts an action on PDGF-BB-induced proliferation via cAMP/PKA/CREB mechanism. SAA blunts PDGF-BB-induced human umbilical artery smooth muscle cell (hUASMC) proliferation via p21 induction, as evidenced by its increased mRNA and protein expression levels. The SAA-induced upregulation of p21 involves the cAMP/PKA signaling pathway; a cAMP analog mimicked the effects of SAA and a specific cAMP/PKA inhibitor opposed these effects. SAA also activated CREB, including phosphorylation at Ser133, and induced its nuclear translocation. Deletion and mutational analysis of p21 promoters, co-immunoprecipitation, and western blot analysis showed that CRE is essential for SAA-induced p21 protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmids into hUASMCs attenuated SAA-stimulated p21 expression. SAA upregulated p21 expression and activated CREB in the neointima of balloon-injured arteries in vivo. Our results indicate that SAA promotes p21 expression in SMCs through the cAMP/PKA/CREB signaling cascade in vitro and prevents injury-induced neointimal hyperplasia.

  4. Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells.

    PubMed

    Kim, Hyung Gyun; Jin, Sun Woo; Kim, Yong An; Khanal, Tilak; Lee, Gi Ho; Kim, Se Jong; Rhee, Sang Dal; Chung, Young Chul; Hwang, Young Jung; Jeong, Tae Cheon; Jeong, Hye Gwang

    2017-08-01

    Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E2. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. MicroRNA-34b promoter hypermethylation induces CREB overexpression and contributes to myeloid transformation

    PubMed Central

    Pigazzi, Martina; Manara, Elena; Bresolin, Silvia; Tregnago, Claudia; Beghin, Alessandra; Baron, Emma; Giarin, Emanuela; Cho, Er-Chieh; Masetti, Riccardo; Rao, Dinesh S.; Sakamoto, Kathleen M.; Basso, Giuseppe

    2013-01-01

    MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and

  6. Cannabinoid receptor type 2 agonist attenuates apoptosis by activation of phosphorylated CREB-Bcl-2 pathway after subarachnoid hemorrhage in rats.

    PubMed

    Fujii, Mutsumi; Sherchan, Prativa; Soejima, Yoshiteru; Hasegawa, Yu; Flores, Jerry; Doycheva, Desislava; Zhang, John H

    2014-11-01

    Early brain injury (EBI) which comprises of vasogenic edema and apoptotic cell death is an important component of subarachnoid hemorrhage (SAH) pathophysiology. This study evaluated whether cannabinoid receptor type 2 (CB2R) agonist, JWH133, attenuates EBI after SAH and whether CB2R stimulation reduces pro-apoptotic caspase-3 via up-regulation of cAMP response element-binding protein (CREB)-Bcl-2 signaling pathway. Male Sprague-Dawley rats (n=123) were subjected to SAH by endovascular perforation. Rats received vehicle or JWH133 at 1h after SAH. Neurological deficits and brain water content were evaluated at 24h after SAH. Western blot was performed to quantify phosphorylated CREB (pCREB), Bcl-2, and cleaved caspase-3 levels. Neuronal cell death was evaluated with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Additionally, CREB siRNA was administered to manipulate the proposed pathway. JWH133 (1.0mg/kg) improved neurological deficits and reduced brain water content in left hemisphere 24h after SAH. JWH133 significantly increased activated CREB (pCREB) and Bcl-2 levels and significantly decreased cleaved caspase-3 levels in left hemisphere 24h after SAH. CREB siRNA reversed the effects of treatment. TUNEL positive neurons in the cortex were reduced with JWH133 treatment. Thus, CB2R stimulation attenuated EBI after SAH possibly through activation of pCREB-Bcl-2 pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Chronic nicotine treatment reverses hypothyroidism-induced impairment of L-LTP induction phase: critical role of CREB.

    PubMed

    Alzoubi, K H; Alkadhi, K A

    2014-06-01

    We have previously shown that adult onset hypothyroidism impairs late-phase long-term potentiation (L-LTP) and reduces basal protein levels of cyclic-AMP response element binding protein (CREB), mutagen-activated protein kinase (MAPKp42/44), and calcium calmodulin kinase IV (CaMKIV) in area Cornu Ammonis 1 (CA1) of the hippocampus. These changes were reversed by chronic nicotine treatment. In the present study, levels of signaling molecules important for L-LTP were determined in CA1 area of the hippocampus during the induction phase. Standard multiple high-frequency stimulation (MHFS) was used to evoke L-LTP in the CA1 area of the hippocampus of hypothyroid, nicotine-treated hypothyroid, nicotine, and sham control anaesthetized adult rats. Chronic nicotine treatment reversed hypothyroidism-induced impairment of L-LTP at the induction phase. Five minutes after MHFS, Western blotting showed an increase in the levels of P-CREB, and P-MAPKp42/44 in sham-operated control, nicotine, and nicotine-treated hypothyroid animals, but not in hypothyroid animals. The protein levels of total CREB, total MAPK p42/44, BDNF, and CaMKIV were not altered in all groups 5 min after MHFS. Therefore, normalized phosphorylation of essential kinases such as P-CREB and P-MAPK p42/44 in the CA1 area of nicotine-treated hypothyroid animals plays a crucial role in nicotine-induced rescue of L-LTP induction during hypothyroidism.

  8. Sleep deprivation prevents stimulation-induced increases of levels of P-CREB and BDNF: protection by caffeine.

    PubMed

    Alhaider, Ibrahim A; Aleisa, Abdulaziz M; Tran, Trinh T; Alkadhi, Karim A

    2011-04-01

    It is well known that caffeine and sleep deprivation have opposing effects on learning and memory; therefore, this study was undertaken to determine the effects of chronic (4wks) caffeine treatment (0.3g/l in drinking water) on long-term memory deficit associated with 24h sleep deprivation. Animals were sleep deprived using the modified multiple platform method. The results showed that chronic caffeine treatment prevented the impairment of long-term memory as measured by performance in the radial arm water maze task and normalized L-LTP in area CA1 of the hippocampi of sleep-deprived anesthetized rats. Sleep deprivation prevents the high frequency stimulation-induced increases in the levels of phosphorylated-cAMP response element binding protein (P-CREB) and brain-derived neurotrophic factor (BDNF) seen during the expression of late phase long-term potentiation (L-LTP). However, chronic caffeine treatment prevented the effect of sleep-deprivation on the stimulated levels of P-CREB and BDNF. The results suggest that chronic caffeine treatment may protect the sleep-deprived brain probably by preserving the levels of P-CREB and BDNF.

  9. Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways.

    PubMed

    Bitner, Robert S; Bunnelle, William H; Anderson, David J; Briggs, Clark A; Buccafusco, Jerry; Curzon, Peter; Decker, Michael W; Frost, Jennifer M; Gronlien, Jens Halvard; Gubbins, Earl; Li, Jinhe; Malysz, John; Markosyan, Stella; Marsh, Kennan; Meyer, Michael D; Nikkel, Arthur L; Radek, Richard J; Robb, Holly M; Timmermann, Daniel; Sullivan, James P; Gopalakrishnan, Murali

    2007-09-26

    The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

  10. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  11. Early induction of CREB activation and CREB-regulating signalling by antidepressants.

    PubMed

    Tardito, Daniela; Musazzi, Laura; Tiraboschi, Ettore; Mallei, Alessandra; Racagni, Giorgio; Popoli, Maurizio

    2009-11-01

    Converging evidence points to adaptive changes in neuroplasticity and gene expression as mediators of therapeutic action of antidepressants. Activation of cAMP response-element binding protein (CREB) and CREB-regulating signalling are considered main effectors in these mechanisms. We analysed the temporal profile of intracellular changes induced by antidepressants, by measuring activation of major CREB-regulating signalling cascades and activation (Ser133 phosphorylation) of CREB. The main aims of the study were to investigate how these different variables are modulated with time, whether stronger activation of signalling cascades corresponds to stronger activation of CREB, and whether these changes are different in distinct brain areas. Rat groups were treated for 1, 2 or 3 wk with the antidepressants fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Activation of CREB and major effectors in signalling cascades were analysed by Western blot analysis with phospho-antibodies, in nuclear and cytosolic fractions from hippocampus and prefrontal/frontal cortex (P/FC). Surprisingly, CREB activation was already maximal after 1-wk treatment. In hippocampus early and stronger CREB activation was consistent with early and stronger activation of signalling. For both drugs, the profile of activation in P/FC was different from that observed in hippocampus. The results also showed that, contrary to the activatory role of MAP-ERKs and CaM kinase IV, nuclear alphaCaM kinase II was inactivated in parallel with activation of CREB.

  12. Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1α in transactivating murine neuroblastic glucose transporter

    PubMed Central

    Thamotharan, Shanthie; Raychaudhuri, Nupur; Tomi, Masatoshi; Shin, Bo-Chul

    2013-01-01

    We have shown in vitro a hypoxia-induced time-dependent increase in facilitative glucose transporter isoform 3 (GLUT3) expression in N2A murine neuroblasts. This increase in GLUT3 expression is partially reliant on a transcriptional increase noted in actinomycin D and cycloheximide pretreatment experiments. Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to −857- to −573-bp and −203- to −177-bp regions. Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1α and p-Creb binding to HRE (−828 to −824 bp) and AP-1 (−187 to −180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays. In addition, the interaction of CBP with Creb and HIF-1α and CREST with CBP in hypoxia was detected by coimmunoprecipitation. Furthermore, small interference (si)RNA targeting Creb in these cells decreased endogenous Creb concentrations that reduced by twofold hypoxia-induced glut3 gene transcription. Thus, in N2A neuroblasts, phosphorylated HIF-1α and Creb mediated the hypoxia-induced increase in glut3 transcription. Coactivation by the Ca++-dependent CREST and CBP proteins may enhance cross-talk between p-Creb-AP-1 and HIF-1α/HRE of the glut3 gene. Collectively, these processes can facilitate an adaptive response to hypoxic energy depletion targeted at enhancing glucose transport and minimizing injury while fueling the proliferative potential of neuroblasts. PMID:23321477

  13. Viral vector induction of CREB expression in the periaqueductal gray induces a predator stress-like pattern of changes in pCREB expression, neuroplasticity, and anxiety in rodents.

    PubMed

    Adamec, Robert; Berton, Olivier; Abdul Razek, Waleed

    2009-01-01

    Predator stress is lastingly anxiogenic. Phosphorylation of CREB to pCREB (phosphorylated cyclic AMP response element binding protein) is increased after predator stress in fear circuitry, including in the right lateral column of the PAG (periaqueductal gray). Predator stress also potentiates right but not left CeA-PAG (central amygdala-PAG) transmission up to 12 days after stress. The present study explored the functional significance of pCREB changes by increasing CREB expression in non-predator stressed rats through viral vectoring, and assessing the behavioral, electrophysiological and pCREB expression changes in comparison with handled and predator stressed controls. Increasing CREB expression in right PAG was anxiogenic in the elevated plus maze, had no effect on risk assessment, and increased acoustic startle response while delaying startle habituation. Potentiation of the right but not left CeA-PAG pathway was also observed. pCREB expression was slightly elevated in the right lateral column of the PAG, while the dorsal and ventral columns were not affected. The findings of this study suggest that by increasing CREB and pCREB in the right lateral PAG, it is possible to produce rats that exhibit behavioral, brain, and molecular changes that closely resemble those seen in predator stressed rats.

  14. Viral Vector Induction of CREB Expression in the Periaqueductal Gray Induces a Predator Stress-Like Pattern of Changes in pCREB Expression, Neuroplasticity, and Anxiety in Rodents

    PubMed Central

    Adamec, Robert; Berton, Olivier; Abdul Razek, Waleed

    2009-01-01

    Predator stress is lastingly anxiogenic. Phosphorylation of CREB to pCREB (phosphorylated cyclic AMP response element binding protein) is increased after predator stress in fear circuitry, including in the right lateral column of the PAG (periaqueductal gray). Predator stress also potentiates right but not left CeA-PAG (central amygdala-PAG) transmission up to 12 days after stress. The present study explored the functional significance of pCREB changes by increasing CREB expression in non-predator stressed rats through viral vectoring, and assessing the behavioral, electrophysiological and pCREB expression changes in comparison with handled and predator stressed controls. Increasing CREB expression in right PAG was anxiogenic in the elevated plus maze, had no effect on risk assessment, and increased acoustic startle response while delaying startle habituation. Potentiation of the right but not left CeA-PAG pathway was also observed. pCREB expression was slightly elevated in the right lateral column of the PAG, while the dorsal and ventral columns were not affected. The findings of this study suggest that by increasing CREB and pCREB in the right lateral PAG, it is possible to produce rats that exhibit behavioral, brain, and molecular changes that closely resemble those seen in predator stressed rats. PMID:19360104

  15. Spatial Memory in the Morris Water Maze and Activation of Cyclic AMP Response Element-Binding (CREB) Protein within the Mouse Hippocampus

    ERIC Educational Resources Information Center

    Porte, Yves; Buhot, Marie Christine; Mons, Nicole E.

    2008-01-01

    We investigated the spatio-temporal dynamics of learning-induced cAMP response element-binding protein activation/phosphorylation (pCREB) in mice trained in a spatial reference memory task in the water maze. Using immunohistochemistry, we examined pCREB immunoreactivity (pCREB-ir) in hippocampal CA1 and CA3 and related brain structures. During the…

  16. Spatial Memory in the Morris Water Maze and Activation of Cyclic AMP Response Element-Binding (CREB) Protein within the Mouse Hippocampus

    ERIC Educational Resources Information Center

    Porte, Yves; Buhot, Marie Christine; Mons, Nicole E.

    2008-01-01

    We investigated the spatio-temporal dynamics of learning-induced cAMP response element-binding protein activation/phosphorylation (pCREB) in mice trained in a spatial reference memory task in the water maze. Using immunohistochemistry, we examined pCREB immunoreactivity (pCREB-ir) in hippocampal CA1 and CA3 and related brain structures. During the…

  17. Changes in the levels and phosphorylation status of Akt, AMPK, CREB, and FoxO1 in hypothalamus of rainbow trout under conditions of enhanced glucosensing activity.

    PubMed

    Otero-Rodiño, Cristina; Velasco, Cristina; Álvarez-Otero, Rosa; López-Patiño, Marcos A; Míguez, Jesús M; Soengas, José L

    2017-10-02

    There is no available information in fish about mechanisms linking glucosensing activation and changes in the expression of brain neuropeptides controlling food intake. Therefore, we assessed in rainbow trout hypothalamus the effects of raised levels of glucose on the levels and phosphorylation status of two transcription factors, FoxO1 and CREB, possibly involved in linking those processes. Moreover, we also aimed to assess the changes in the levels and phosphorylation status of two proteins possibly involved in the modulation of these transcription factors such as Akt and AMPK. Therefore, we evaluated in pools of hypothalamus incubated for 3h and 6h at 15 °C in modified Hanks' medium containing 2, 4, or 8 mM D-glucose the response of parameters related to glucosensing mechanisms, neuropeptide expression, and levels and phosphorylation status of proteins of interest. The activation of hypothalamic glucosensing systems and the concomitant enhanced anorectic potential occurred in parallel with activation of Akt and inhibition of AMPK. The changes in these proteins would relate to neuropeptide expression through changes in the levels and phosphorylation status of transcription factors under their control, such as CREB and FoxO1, which displayed inhibitory (CREB) or activatory (FoxO1) responses to increased glucose. © 2017. Published by The Company of Biologists Ltd.

  18. Resveratrol abrogates lipopolysaccharide-induced depressive-like behavior, neuroinflammatory response, and CREB/BDNF signaling in mice.

    PubMed

    Ge, Li; Liu, Liwei; Liu, Hansen; Liu, Song; Xue, Hao; Wang, Xueer; Yuan, Lin; Wang, Zhen; Liu, Dexiang

    2015-12-05

    Current evidence supports that depression is accompanied by the activation of the inflammatory-response system, and overproduction of pro-inflammatory cytokines may play a role in the pathophysiology of depressive disorders. Resveratrol has anti-inflammatory, antioxidant and anti-depressant-like properties. Using an animal model of depression induced by a single administration of lipopolysaccharide (LPS), the present study investigated the effects of resveratrol on LPS-induced depressive-like behavior and inflammatory-response in adult mice. Our results showed that pretreatment with resveratrol (80mg/kg, i.p.) for 7 consecutive days reversed LPS-increased the immobility time in the forced swimming test and tail suspension test, and LPS-reduced sucrose preference test. Moreover, the antidepressant action of resveratrol was paralleled by significantly reducing the expression levels of pro-inflammatory cytokines, and up-regulating phosphorylated cAMP response-element-binding protein (pCREB)/brain-derived neurotrophic factor (BDNF) expression in prefrontal cortex (PFC) and hippocampus. In addition, resveratrol ameliorated LPS-induced NF-κB activation in the PFC and hippocampus. The results demonstrate that resveratrol may be an effective therapeutic agent for LPS-induced depressive-like behavior, partially due to its anti-inflammatory aptitude and by modulating pCREB and BDNF expression in the brain region of mice.

  19. [Immunohistochemical detection of transcription factors CREB and c-Fos activation in the land snail nervous system induced by pentylenetetrazole].

    PubMed

    Efimova, O I; Ierusalimskiĭ, V N; Anokhin, K V; Balaban, P M

    2006-01-01

    Phosphorylation of transcription factor CREB and expression of immediate early gene c-fos play a key role in molecular mechanisms of long-term neuronal plasticity in the vertebrate brain. Here, we have defined the procedure of immunohistochemical detection of pCREB and c-Fos in the nervous system of the land snail Helix lucorum (Pulmonata: Stylommatophora) and have shown its activation after the convulsant pentylenetetrazole injection. Baseline pCREB1 and c-Fos levels in the intact snail nervous system appeared to be low. In contrast, injection of pentylenetetrazole (600 mg/kg) produced a rapid induction of CREB phosphorylation and c-Fos expression in a wide range of neurons including a number of identified cells. Double immunofluorescence for pCREB and c-Fos showed that c-Fos was always colocalized with pCREB-immunoreactivity, although the latter had a broader pattern. The results suggest that transcription factors CREB and c-Fos can be used as molecular markers for mapping long-term neuronal plasticity in molluscan nervous system.

  20. Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse.

    PubMed

    Lim, Soonmin; Moon, Minho; Oh, Hyein; Kim, Hyo Geun; Kim, Sun Yeou; Oh, Myung Sook

    2014-10-01

    Ginger (the rhizome of Zingiber officinale Roscoe) has been used worldwide for many centuries in cooking and for treatment of several diseases. The main pharmacological properties of ginger include anti-inflammatory, antihyperglycemic, antiarthritic, antiemetic and neuroprotective actions. Recent studies demonstrated that ginger significantly enhances cognitive function in various cognitive disorders as well as in healthy brain. However, the biochemical mechanisms underlying the ginger-mediated enhancement of cognition have not yet been studied in normal or diseased brain. In the present study, we assessed the memory-enhancing effects of dried ginger extract (GE) in a model of scopolamine-induced memory deficits and in normal animals by performing a novel object recognition test. We found that GE administration significantly improved the ability of mice to recognize novel objects, indicating improvements in learning and memory. Furthermore, to elucidate the mechanisms of GE-mediated cognitive enhancement, we focused on nerve growth factor (NGF)-induced signaling pathways. NGF enzyme-linked immunosorbent assay analysis revealed that GE administration led to elevated NGF levels in both the mouse hippocampus and rat glioma C6 cells. GE administration also resulted in phosphorylation of extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), as revealed by Western blotting analysis. Neutralization of NGF with a specific NGF antibody inhibited GE-triggered activation of ERK and CREB in the hippocampus. Also, GE treatment significantly increased pre- and postsynaptic markers, synaptophysin and PSD-95, which are related to synapse formation in the brain. These data suggest that GE has a synaptogenic effect via NGF-induced ERK/CREB activation, resulting in memory enhancement. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

    SciTech Connect

    Corona, Giulia; Deiana, Monica; Incani, Alessandra; Vauzour, David; Assunta Dessi, M.; Spencer, Jeremy P.E.

    2007-10-26

    We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 {mu}g/ml) the number of cells in the G2/M phase increased to 51.82 {+-} 2.69% relative to control cells (15.1 {+-} 2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 {mu}g/ml) to exert rapid inhibition of p38 (38.7 {+-} 4.7%) and CREB (28.6 {+-} 5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9 {+-} 9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.

  2. Dynorphin up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR is associated with increased CREB (Ser 133) phosphorylation, but is independent of BDNF/TrkB signaling pathways

    PubMed Central

    Rittase, W. Bradley; Dong, Yu; Barksdale, DaRel; Galdzicki, Zygmunt; Bausch, Suzanne B.

    2014-01-01

    Emerging evidence suggests that neuronal responses to N-methyl-d-aspartate (NMDAR) activation/inactivation are influenced by subunit composition. For example, activation of synaptic NMDAR (comprised of GluN2A > GluN2B) phosphorylates cAMP-response-element-binding protein (CREB) at Ser 133, induces BDNF expression and promotes neuronal survival. Activation of extrasynaptic NMDAR (comprised of GluN2B>GluN2), dephosphorylates CREB (Ser 133), reduces BDNF expression and triggers neuronal death. These results led us to hypothesize that chronic inhibition of GluN2B-containing NMDAR would increase CREB (Ser 133) phosphorylation, increase BDNF levels and subsequently alter downstream dynorphin (DYN) and neuropeptide Y (NPY) expression. We focused on DYN and NPY because these neuropeptides can decrease excitatory neurotransmission and seizure occurrence and we reported previously that seizure-like events are reduced following chronic treatment with GluN2B antagonists. Consistent with our hypothesis, chronic treatment (17-21 days) of hippocampal slice cultures with the GluN2B-selective antagonists ifenprodil or Ro25,6981 increased both CREB (Ser 133) phosphorylation and granule cell mossy fiber pathway DYN expression. Similar treatment with the non-subtype-selective NMDAR antagonists D-APV or memantine had no significant effect on either CREB (Ser 133) phosphorylation or DYN expression. In contrast to our hypothesis, BDNF levels were decreased following chronic treatment with Ro25,6981, but not ifenprodil, D-APV or memantine. Blockade of BDNF actions and TrkB activation did not significantly augment hilar DYN expression in vehicle-treated cultures and had no effect in Ro25,6981 treated cultures. These finding suggest that chronic exposure to GluN2B-selective NMDAR antagonists increased DYN expression through a putatively pCREB-dependent, but BDNF/TrkB-independent mechanism. PMID:24769103

  3. Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells

    PubMed Central

    Huang, Hao; Hong, Qian; Tan, Hong-ling; Xiao, Cheng-rong; Gao, Yue

    2016-01-01

    Aim: Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells. Methods: PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA. Results: Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10–40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2. Conclusion: The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS-induced

  4. CREB Mediates Prostaglandin F2α-Induced MUC5AC Overexpression

    PubMed Central

    Chung, Wen-Cheng; Ryu, Seung-Hee; Sun, Hongxia; Zeldin, Darryl C.; Koo, Ja Seok

    2009-01-01

    Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are prostaglandin (PG) E2 and F2α. PGE2-induced mucin production has been well studied, but the effect of PGF2α on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF2α on MUC5AC production, we investigated the signal transduction of PGF2α associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF2α induces MUC5AC overproduction via a signaling cascade involving protein kinase C, extracellular signal-regulated kinase, p90 ribosomal S6 protein kinase, and cAMP response element binding protein (CREB). The regulation of PGF2α-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF2α receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE2 and other inflammatory mediators, our findings have important clinical implication for the management of airway mucus hypersecretion. PMID:19201889

  5. cAMP-dependent protein kinase type I regulates ethanol-induced cAMP response element-mediated gene expression via activation of CREB-binding protein and inhibition of MAPK.

    PubMed

    Constantinescu, Anastasia; Wu, Meiye; Asher, Orna; Diamond, Ivan

    2004-10-08

    We have shown that the two types of cAMP-dependent protein kinase (PKA) in NG108-15 cells differentially mediate forskolin- and ethanol-induced cAMP response element (CRE)-binding protein (CREB) phosphorylation and CRE-mediated gene transcription. Activated type II PKA is translocated into the nucleus where it phosphorylates CREB. By contrast, activated type I PKA does not translocate to the nucleus but is required for CRE-mediated gene transcription by inducing the activation of other transcription cofactors such as CREB-binding protein (CBP). We show here that CBP is required for forskolin- and ethanol-induced CRE-mediated gene expression. Forskolin- and ethanol-induced CBP phosphorylation, demonstrable at 10 min, persists up to 24 h. CBP phosphorylation requires type I PKA but not type II PKA. In NG108-15 cells, ethanol and forskolin activation of type I PKA also inhibits several components of the MAPK pathway including B-Raf kinase, ERK1/2, and p90RSK phosphorylation. As a result, unphosphorylated p90RSK no longer binds to nor inhibits CBP. Moreover, MEK inhibition by PD98059 induces a significant increase of CRE-mediated gene activation. Taken together, our findings suggest that inhibition of the MAPK pathway enhances cAMP-dependent gene activation during exposure of NG108-15 cells to ethanol. This mechanism appears to involve type I PKA-dependent phosphorylation of CBP and inhibition of MEK-dependent phosphorylation of p90RSK. Under these conditions p90RSK is no longer bound to CBP, thereby promoting CBP-dependent CREB-mediated gene expression.

  6. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

    SciTech Connect

    Lee, Guan-Lin; Wu, Jing-Yiing; Yeh, Chang-Ching; Kuo, Cheng-Chin

    2016-05-13

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

  7. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration.

    PubMed

    Lee, Guan-Lin; Wu, Jing-Yiing; Yeh, Chang-Ching; Kuo, Cheng-Chin

    2016-05-13

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis.

  8. Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice

    PubMed Central

    Cho, Woo-Hyun; Han, Jung-Soo

    2016-01-01

    Flexibility in using different learning strategies was assessed in two different inbred strains of mice, the C57BL/6 and DBA/2 strains. Mice were trained sequentially in two different Morris water maze protocols that tested their ability to switch their learning strategy to complete a new task after first being trained in a different task. Training consisted either of visible platform trials (cued training) followed by subsequent hidden platform trials (place training) or the reverse sequence (place training followed by cued training). Both strains of mice showed equivalent performance in the type of training (cued or place) that they received first. However, C57BL/6 mice showed significantly better performances than DBA/2 mice following the switch in training protocols, irrespective of the order of training. After completion of the switched training session, levels of cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) were measured in the hippocampus, striatum and prefrontal cortex of the mice. Prefrontal cortical and hippocampal pCREB levels differed by strain, with higher levels found in C57BL/6 mice than in DBA/2 mice. No strain differences were observed in the medial or lateral region of the dorsal striatum. These findings indicate that the engagement (i.e., CREB signaling) of relevant neural structures may vary by the specific demands of the learning strategy, and this is closely tied to differences in the flexibility of C57BL/6 and DBA/2 mice to switch their learning strategies when given a new task. PMID:27695401

  9. Differences in the Flexibility of Switching Learning Strategies and CREB Phosphorylation Levels in Prefrontal Cortex, Dorsal Striatum and Hippocampus in Two Inbred Strains of Mice.

    PubMed

    Cho, Woo-Hyun; Han, Jung-Soo

    2016-01-01

    Flexibility in using different learning strategies was assessed in two different inbred strains of mice, the C57BL/6 and DBA/2 strains. Mice were trained sequentially in two different Morris water maze protocols that tested their ability to switch their learning strategy to complete a new task after first being trained in a different task. Training consisted either of visible platform trials (cued training) followed by subsequent hidden platform trials (place training) or the reverse sequence (place training followed by cued training). Both strains of mice showed equivalent performance in the type of training (cued or place) that they received first. However, C57BL/6 mice showed significantly better performances than DBA/2 mice following the switch in training protocols, irrespective of the order of training. After completion of the switched training session, levels of cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) were measured in the hippocampus, striatum and prefrontal cortex of the mice. Prefrontal cortical and hippocampal pCREB levels differed by strain, with higher levels found in C57BL/6 mice than in DBA/2 mice. No strain differences were observed in the medial or lateral region of the dorsal striatum. These findings indicate that the engagement (i.e., CREB signaling) of relevant neural structures may vary by the specific demands of the learning strategy, and this is closely tied to differences in the flexibility of C57BL/6 and DBA/2 mice to switch their learning strategies when given a new task.

  10. Dioscin Derived from Solanum melongena L. "Usukawamarunasu" Attenuates α-MSH-Induced Melanogenesis in B16 Murine Melanoma Cells via Downregulation of Phospho-CREB and MITF.

    PubMed

    Nishina, Atsuyoshi; Ebina, Kodai; Ukiya, Motohiko; Fukatsu, Makoto; Koketsu, Mamoru; Ninomiya, Masayuki; Sato, Daisuke; Kimura, Hirokazu

    2015-10-01

    This study aimed to chemically isolate and explore an antimelanogenesis inducer in extracts of Solanum melongena L. "Usukawamarunasu" eggplant. We successfully identified dioscin ([25R]-Spirost-5-en-3β-yl) 2-O-(6-deoxy-α-L-mannopyranosyl) - 4-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside] in the plant, and examined the effects of α-melanocyte-stimulating hormone (MSH)-induced melanogenesis in B16 murine melanoma cells by this plant-derived dioscin. Immunoblot analysis suggested that dioscin reduced the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, resulting in inhibition of intracellular production of melanin. In addition, dioscin caused reduction of phosphorylated cAMP-responsive element binding protein 1 transcription factors (CREB), which led to a reduction of microphthalmia-related transcription factor (MITF) in α-MSH-stimulated cells, but did not affect phosphorylation of extracellular signal-regulated kinase. Furthermore, dioscin significantly downregulated the expression of tyrosinase, TRP-1, and TRP-2, which led to the reduction of α-MSH-induced melanogenesis in B16 cells. These results suggest that dioscin may decrease the level of MITF via inhibition of phosphorylation of CREB in α-MSH-induced melanogenesis in B16 cells. © 2015 Institute of Food Technologists®

  11. Interplay of CREB and ATF2 in Ionizing Radiation-Induced Neuroendocrine Differentiation of Prostate Cancer Cells

    DTIC Science & Technology

    2012-06-01

    that contributes to IR- induced phos- phorylation of CREB. It will be interesting to determine whether this effect is independent of or dependent on...S. (2010) Growth factor stimulation induces cell survival by c- Jun. ATF2- dependent activation of Bcl-XL. J. Biol. Chem. 285, 23096–23104 7. Shimizu...08-1-0394 TITLE: Interplay of CREB and ATF2 in Ionizing Radiation- Induced Neuroendocrine Differentiation of Prostate Cancer Cells

  12. Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

    PubMed Central

    Mair, William; Morantte, Ianessa; Rodrigues, Ana P. C.; Manning, Gerard; Montminy, Marc; Shaw, Reuben J.; Dillin, Andrew

    2011-01-01

    Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans1,2 and both have been implicated as therapeutic targets for age-related pathology in mammals3–5. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs)6 are a family of cofactors involved in diverse physiological processes including energy homeostasis7–9, cancer10 and endoplasmic reticulum stress11. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity. PMID:21331044

  13. CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

    PubMed Central

    Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

    2014-01-01

    It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

  14. Hydrocortisone-induced parkin prevents dopaminergic cell death via CREB pathway in Parkinson's disease model.

    PubMed

    Ham, Sangwoo; Lee, Yun-Il; Jo, Minkyung; Kim, Hyojung; Kang, Hojin; Jo, Areum; Lee, Gum Hwa; Mo, Yun Jeong; Park, Sang Chul; Lee, Yun Song; Shin, Joo-Ho; Lee, Yunjong

    2017-04-03

    Dysfunctional parkin due to mutations or post-translational modifications contributes to dopaminergic neurodegeneration in Parkinson's disease (PD). Overexpression of parkin provides protection against cellular stresses and prevents dopamine cell loss in several PD animal models. Here we performed an unbiased high-throughput luciferase screening to identify chemicals that can increase parkin expression. Among promising parkin inducers, hydrocortisone possessed the most favorable profiles including parkin induction ability, cell protection ability, and physicochemical property of absorption, distribution, metabolism, and excretion (ADME) without inducing endoplasmic reticulum stress. We found that hydrocortisone-induced parkin expression was accountable for cell protection against oxidative stress. Hydrocortisone-activated parkin expression was mediated by CREB pathway since gRNA to CREB abolished hydrocortisone's ability to induce parkin. Finally, hydrocortisone treatment in mice increased brain parkin levels and prevented 6-hydroxy dopamine induced dopamine cell loss when assessed at 4 days after the toxin's injection. Our results showed that hydrocortisone could stimulate parkin expression via CREB pathway and the induced parkin expression was accountable for its neuroprotective effect. Since glucocorticoid is a physiological hormone, maintaining optimal levels of glucocorticoid might be a potential therapeutic or preventive strategy for Parkinson's disease.

  15. Topiramate Confers Neuroprotection Against Methylphenidate-Induced Neurodegeneration in Dentate Gyrus and CA1 Regions of Hippocampus via CREB/BDNF Pathway in Rats.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh; Abdollahi, Mohammad; Heidari, Mansour; Madjd, Zahra

    2017-04-01

    Methylphenidate (MPH) abuse can cause serious neurological damages. The neuroprotective effects of topiramate (TPM) have been reported already, but its mechanism of action still remains unclear. The current study evaluates in vivo role of CREB/BDNF in TPM protection of the rat hippocampal cells from methylphenidate-induced apoptosis, oxidative stress, and inflammation. A total of 60 adult male rats were divided into six groups. Groups 1 and 2 received normal saline (0.7 ml/rat) and MPH (10 mg/kg) respectively for 14 days. Groups 3 and 4 were concurrently treated with MPH (10 mg/kg) and TPM 50 and 100 mg/kg respectively for 14 days. Groups 5 and 6 were treated with 50 and 100 mg/kg TPM only respectively. After drug administration, open field test (OFT) was used to investigate motor activity. The hippocampus was then isolated and the apoptotic, antiapoptotic, oxidative, antioxidant, and inflammatory factors were measured. Expression of the total and phosphorylated CREB and BDNF in gene and protein levels, and gene expression of Ak1, CaMK4, MAPK3, PKA, and c-Fos levels were also measured. MPH significantly decreased motor activity in OFT. TPM (50 and 100 mg/kg) decreased MPH-induced motor activity disturbance. Additionally, MPH significantly increased Bax protein level, CaMK4 gene expression, lipid peroxidation, catalase activity, mitochondrial GSH, IL-1β, and TNF-α levels in isolated hippocampal cells. Also CREB, in total and phosphorylated forms, BDNF and Bcl-2 protein levels, Ak1, MAPK3, PKA and c-Fos gene expression, superoxide dismutase, glutathione peroxidase, and glutathione reductase activities decreased significantly by MPH. TPM (50 and 100 mg/kg), both in the presence and absence of MPH, attenuated the effects of MPH. Immunohistochemistry data showed that TPM increased localization of the total and phosphorylated forms of CREB in dentate gyrus (DG) and CA1 areas of the hippocampus. It seems that TPM can be used as a neuroprotective agent against

  16. Protein phosphorylation cascades associated with methamphetamine-induced glial activation.

    PubMed

    Hebert, M A; O'Callaghan, J P

    2000-09-01

    Reactive gliosis is the most prominent response to diverse forms of central nervous system (CNS) injury. The signaling events that mediate this characteristic response to neural injury are under intense investigation. Several studies have demonstrated the activation of phosphoproteins within the mitogen-activated protein kinase (MAPK) and Janus kinase (JAK) pathways following neural insult. These signaling pathways may be involved or responsible for the glial response following injury, by virtue of their ability to phosphorylate and dynamically regulate the activity of various transcription factors. This study sought to delineate, in vivo, the relative contribution of MAPK- and JAK-signaling components to reactive gliosis as measured by induction of glial-fibrillary acidic protein (GFAP), following chemical-induced neural damage. At time points (6, 24, and 48 h) following methamphetamine (METH, 10 mg/kg x 4, s.c.) administration, female C57BL/6J mice were sacrificed by focused microwave irradiation, a technique that preserves steady-state phosphorylation. Striatal (target) and nontarget (hippocampus) homogenates were assayed for METH-induced changes in markers of dopamine (DA) neuron integrity as well as differences in the levels of activated phosphoproteins. GFAP upregulation occurred as early as 6 h, reaching a threefold induction 48 h following METH exposure. Neurotoxicant-induced reductions in striatal levels of DA and tyrosine hydroxylase (TH) paralleled the temporal profile of GFAP induction. Blots of striatal homogenates, probed with phosphorylation-state specific antibodies, demonstrated significant changes in activated forms of extracellular-regulated kinase 1/2 (ERK 1/2), c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), MAPK/ERK kinase (MEK1/2), 70-kDa ribosomal S6 kinase (p70 S6), cAMP responsive element binding protein (CREB), and signal transducer and activator of transcription 3 (STAT3). MAPK-related phosphoproteins exhibited an

  17. Scopolamine induced amnesia is reversed by Bacopa monniera through participation of kinase-CREB pathway.

    PubMed

    Saraf, Manish Kumar; Anand, Akshay; Prabhakar, Sudesh

    2010-02-01

    Scopolamine, an anticholinergic drug, is reported to produce amnesia by interference of long term potentiation and has been used for discerning the efficacy of various antiamnesic drugs. The intoxication with anticholinergics and benzodiazepines tend to produce neurodegeneration which cause memory deficits. Our earlier reports have shown the antiamnesic drug, B. monniera to be capable of alleviating diazepam induced memory deficits. We have now tested how scopolamine affects downstream signaling molecules of long term potentiation and if B. monniera can also modulate the scopolamine induced amnesia. We used Morris water maze scale to test the amnesic effect of scopolamine and its reversal by B. monniera. Rota-rod test was used to screen muscle coordination activity of mice before water maze investigations were carried out. The results showed that scopolamine downregulated protein kinase C and iNOS without affecting cAMP, protein kinase A, calmodulin, MAP kinase, nitrite, CREB and pCREB. B. monniera reversed the scopolamine induced amnesia by significantly improving calmodulin and by partially attenuating protein kinase C and pCREB. These observations suggest involvement of calmodulin in evoking antiamnesic effects of B. monniera.

  18. Puerarin protects mouse liver against nickel-induced oxidative stress and inflammation associated with the TLR4/p38/CREB pathway.

    PubMed

    Liu, Chan-Min; Ma, Jie-Qiong; Liu, Si-Si; Feng, Zhao-Jun; Wang, Ai-Min

    2016-01-05

    Nickel (Ni), one of hazardous environmental chemicals, is known to cause liver injury. Accumulating evidence showed that puerarin (PU) possessed comprehensive biological effects. The purpose of the current study was to test the hypothesis that the puerarin protects against enhanced liver injury caused by Ni in mice. ICR mice received intraperitoneally nickel sulfate (20 mg/kg/body weight, daily) for 20 days, and puerarin (200 and 400 mg/kg/body weight) was applied before Ni exposure. The results indicated that puerarin markedly inhibited Ni-induced liver injury, which was characterized by decreased aminotransferase activities and inflammation. Puerarin also inhibited the oxidative stress and decreased the metallothionein (MT) levels. Puerarin decreased the level of pro-inflammatory cytokines TNF-α and IL-6 in livers. Puerarin significantly inhibited the TLR4 activation and p38 MAPK phosphorylation, which in turn inhibited NF-κB activity. Likewise, Ni-induced inflammatory responses were diminished by puerarin as observed by a remarkable reduction in the levels of phosphorylated CREB. Furthermore, puerarin also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) levels in livers. Data from this study suggested that the inhibition of Ni-induced oxidative stress and inflammatory responses by puerarin is due to its ability to modulate the TLR4/p38/CREB signaling pathway.

  19. Isoflurane-induced inactivation of CREB through histone deacetylase 4 is responsible for cognitive impairment in developing brain.

    PubMed

    Sen, Tanusree; Sen, Nilkantha

    2016-12-01

    Anesthetics including isoflurane are known to induce neuronal dysfunction in the developing brain, however, the underlying mechanism is mostly unknown. The transcriptional activation of CREB (cyclic AMP response element binding protein) and the alterations in acetylation of histones modulated by several histone deacetylases such as HDAC4 (histone deacetylase 4) are known to contribute to synaptic plasticity in the brain. Here we have shown that administration of isoflurane (1.4%) for 2h leads to transcriptional inactivation of CREB which results in loss of dendritic outgrowth and decreased expression level of proteins essential for memory and cognitive functions, such as BDNF, and c-fos in the developing brain of mice at postnatal day 7 (PND7). To elucidate the molecular mechanism, we found that exposure to isoflurane leads to an increase in nuclear translocation of HDAC4, which interacts with CREB in the nucleus. This event, in turn, results in a decrease in interaction between an acetyltransferase, CBP, and CREB that ultimately leads to transcriptional inactivation of CREB. As a result, the expression level of BDNF, and c-fos were significantly down-regulated after administration of isoflurane in PND7 brain. Depletion of HDAC4 in PND7 brain rescues the transcriptional activation of CREB along with augmentation in the level of the expression level of BDNF and c-fos. Moreover, administration of lentiviral particles of HDAC4 RNAi in primary neurons rescues neurite outgrowth following isoflurane treatment. Taken together, our study suggests that HDAC4-induced transcriptional inactivation of CREB is responsible for isoflurane-induced cognitive dysfunction in the brain. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. N-palmitoyl serotonin alleviates scopolamine-induced memory impairment via regulation of cholinergic and antioxidant systems, and expression of BDNF and p-CREB in mice.

    PubMed

    Min, A Young; Doo, Choon Nan; Son, Eun Jung; Sung, Nak Yun; Lee, Kun Jong; Sok, Dai-Eun; Kim, Mee Ree

    2015-12-05

    N-Palmitoyl-5-hydroxytryptamines (Pal-5HT), a cannabinoid, has recently been reported to express anti-allergic and anti-inflammatory actions in RBL-2H3 cells, and ameliorate glutamate-induced cytotoxicity in HT-22 cells. In this study, we examined the effect of Pal-5HT on deficits of learning and memory induced by scopolamine in mice. Memory performance was evaluated using Morris water maze test and passive avoidance test. Activities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT), level of oxidative stress markers, and expression of brain-derived neurotrophic factor (BDNF), phosphorylation of cAMP response element-binding protein (p-CREB) were determined. Loss of neuronal cells in hippocampus was evaluated by histological examinations. Pal-5HT significantly improved the amnesia in the behavioral assessment. Pal-5HT regulated cholinergic function by inhibiting scopolamine-induced elevation of AChE activity and decline of ChAT activity. Pal-5HT suppressed oxidative stress by increasing activities of glutathione peroxidase (GPx), glutathione reductase (GR) or NAD(P)H quinine oxidoreductase-1 (NQO-1) and lowering MDA level. Additionally, it prevented against scopolamine-induced expression of iNOS and COX-2. Moreover, Pal-5HT suppressed the death of neuronal cells in CA1 and CA3 regions, while it restored expression of p-CREB and BDNF in hippocampus. Taken together, Pal-5HT is suggested to ameliorate deficits of memory and learning through regulation of cholinergic function, activation of antioxidant systems as well as restoration of BDNF and p-CREB expression. From these, Pal-5HT may be a potential candidate to prevent against neurodegeneration related to the memory deficit.

  1. Immunohistochemical detection of the activation of CREB and c-Fos transcription factors in the nervous system of the terrestrial snail induced by pentylenetetrazole.

    PubMed

    Efimova, O I; Ierusalimskii, V N; Anokhin, K V; Balaban, P M

    2007-11-01

    Phosphorylation of CREB transcription factor and expression of the immediate early gene c-fos in the brains of vertebrates play a key role in the molecular genetic mechanisms of long-term neuronal plasticity. The present study identifies the conditions for immunohistochemical detection of pCREB and c-Fos in the nervous system of the mollusk Helix lucorum (Pulmonata: Stylommatophora); activation of these transcription factors was demonstrated after administration of the convulsive agent pentylenetetrazole. Basal pCREB and c-Fos levels in the central nervous system of intact animals were low. Injection of pentylenetetrazole at a dose of 600 mg/kg evoked characteristic stereotypical motor responses, along with sharp reductions in the phosphorylation of CREB1 and expression of the immediate early gene c-fos, this also occurring in identified neurons. Double immunofluorescent labeling of pCREB and c-Fos showed that expression of c-Fos transcription factor was seen only in pCREB-immunoreactive neurons. These data provide evidence that activation of pCREB and c-Fos transcription factors can be used as molecular markers for mapping the processes of neuronal plasticity in the nervous systems of mollusks.

  2. Unconditioned- and Conditioned- Stimuli Induce Differential Memory Reconsolidation and β-AR-Dependent CREB Activation.

    PubMed

    Huang, Bing; Zhu, Huiwen; Zhou, Yiming; Liu, Xing; Ma, Lan

    2017-01-01

    Consolidated long-term fear memories become labile and reconsolidated upon retrieval by the presentation of conditioned stimulus (CS) or unconditioned stimulus (US). Whether CS-retrieval or US-retrieval will trigger different memory reconsolidation processes is unknown. In this study, we introduced a sequential fear conditioning paradigm in which footshock (FS) was paired with two distinct sounds (CS-A and CS-B). The treatment with propranolol, a β-adrenergic receptor (β-AR) antagonist, after US (FS)-retrieval impaired freezing behavior evoked by either CS-A or CS-B. Betaxolol, a selective β1-AR antagonist, showed similar effects. However, propranolol treatment after retrieval by one CS (e.g., CS-A) only inhibited freezing behavior evoked by the same CS (i.e., CS-A), not the other CS (CS-B). These data suggest that β-AR is critically involved in reconsolidation of fear memory triggered by US- and CS-retrieval, whereas β-AR blockade after US-retrieval disrupts more CS-US associations than CS-retrieval does. Furthermore, significant CREB activation in almost the whole amygdala and hippocampus was observed after US-retrieval, but CS-retrieval only stimulated CREB activation in the lateral amygdala and the CA3 of hippocampus. In addition, propranolol treatment suppressed memory retrieval-induced CREB activation. These data indicate that US-retrieval activates more memory traces than CS-retrieval does, leading to memory reconsolidation of more CS-US associations.

  3. IDENTIFICATION OF CHOLINERGIC AND NONCHOLINERGIC NEURONS IN THE PONS EXPRESSING PHOSPHORYLATED cAMP RESPONSE ELEMENT-BINDING PROTEIN (pCREB) AS A FUNCTION OF RAPID EYE MOVEMENT (REM) SLEEP

    PubMed Central

    DATTA, S.; SIWEK, D. F.; STACK, E. C.

    2009-01-01

    Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of REM sleep. In the present study performed on rats, phosphorylated cAMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high REM sleep (HR, ~27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With high REM sleep, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low REM sleep (LR, ~2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr = 0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr = 0.88), PnO (Rsqr = 0.87), and SubCD (Rsqr = 0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr = 0.70) and DRN (Rsqr = 0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are inactive. More importantly, the regression analysis

  4. miR-134 regulates ischemia/reperfusion injury-induced neuronal cell death by regulating CREB signaling.

    PubMed

    Huang, Weidong; Liu, Xiaobin; Cao, Jie; Meng, Facai; Li, Min; Chen, Bo; Zhang, Jie

    2015-04-01

    microRNA-134 (miR-134) has been reported to be a brain-specific miRNA and is differently expressed in brain tissues subjected to ischemic injury. However, the underlying mechanism of miR-134 in regulating cerebral ischemic injury remains poorly understood. The current study was designed to delineate the molecular basis of miR-134 in regulating cerebral ischemic injury. Using the oxygen-glucose deprivation (OGD) model of hippocampal neuron ischemia in vitro, we found that the overexpression of miR-134 mediated by recombinant adeno-associated virus (AAV) vector infection significantly promoted neuron death induced by OGD/reoxygenation, whereas the inhibition of miR-134 provided protective effects against OGD/reoxygenation-induced cell death. Moreover, cyclic AMP (cAMP) response element-binding protein (CREB) as a putative target of miR-134 was downregulated and upregulated by miR-134 overexpression or inhibition, respectively. The direct interaction between miR-134 and the 3'-untranslated region (UTR) of CREB mRNA was further confirmed by dual-luciferase reporter assay. Overexpression of miR-134 also inhibited the expression of the downstream gene of CREB, including brain-derived neurotrophic factor (BDNF) and the anti-apoptotic gene Bcl-2, whereas the inhibition of miR-134 upregulated the expression of BDNF and Bcl-2 in neurons after OGD/reoxygenation. Notably, the knockdown of CREB by CREB siRNA apparently abrogated the protective effect of anti-miR-134 on OGD/reoxygenation-induced cell death. Taken together, our study suggests that downregulation of miR-134 alleviates ischemic injury through enhancing CREB expression and downstream genes, providing a promising and potential therapeutic target for cerebral ischemic injury.

  5. Chronic Intermittent Hypobaric Hypoxia Pretreatment Ameliorates Ischemia-Induced Cognitive Dysfunction Through Activation of ERK1/2-CREB-BDNF Pathway in Anesthetized Mice.

    PubMed

    Wang, Jintian; Zhang, Shixiao; Ma, Huijuan; Yang, Shijie; Liu, Zhao; Wu, Xiaolei; Wang, Sheng; Zhang, Yi; Liu, Yixian

    2017-02-01

    Chronic intermittent hypobaric hypoxia (CIHH) has protective effects on heart and brain against ischemia injury through mobilizing endogenous adaptive mechanisms. However, whether CIHH prevents against cognitive impairment was not elucidated. The present study aimed to investigate the effect and mechanism of CIHH treatment on ischemia/reperfusion (IR)-induced cognitive dysfunction. Mice were randomly divided into 8 groups: Control, Sham, CIHH (simulating 5000 m high-altitude for 28 days, 6 h per day), IR (three 16-min occlusions of bilateral common carotid arteries interrupted by two 10-min intervals), CIHH + IR, PD98059 (inhibitor of MEK1/2) + CIHH + IR, PD98059 + Sham and PD98059 + IR group. Morris water maze and step-down passive avoidance tests were performed to evaluate the capability of learning and memory 1 month after ischemia. Thionine dyeing was to examine histological manifestations of pyramidal neurons in hippocampus CA1 region. Western blotting assay was for measurement of the protein expressions in ERK1/2-CREB-BDNF signaling pathway. There were a shorter escape latency and a longer percentage of time retaining in the target quadrant in Morris water maze test, fewer times of errors in the step-down avoidance test and a higher neuronal density of the hippocampal CA1 subfield in CIHH + IR group than in IR group. CIHH upregulated the expressions of BDNF, phosphorylated CREB, ERK1/2 and TrkB with or without ischemia. The protective effects of CIHH were abolished by PD98059 administration 15 min before ischemia. CIHH ameliorated ischemia-induced cognitive dysfunction through activation of ERK1/2-CREB-BDNF signaling pathway.

  6. Reversion of BDNF, Akt and CREB in Hippocampus of Chronic Unpredictable Stress Induced Rats: Effects of Phytochemical, Bacopa Monnieri

    PubMed Central

    Hazra, Somoday; Kumar, Sourav; Saha, Goutam Kumar

    2017-01-01

    Objective The aims of the present study were to explore the behavioural effects and to understand the possible mode of action of Bacopa monnieri extract (BME) on chronic unpredictable stress (CUS) induced depressive model and the biochemical alterations such as brain derived neurotrophic factor (BDNF), Akt, cyclic-AMP response element binding (CREB) protein level in the hippocampus of rats. Methods We examined the effects of chronic administration of BME on CUS exposed rats for 28 days. Behavioural changes were assessed by sucrose consumption and open field test to assess the effect of BME on CUS-induced depression. The mechanisms underlying antidepressant like action of BME was further evaluated by measuring levels of BDNF, Akt, and CREB in the hippocampus of rat brain and compared with the standard tricyclic antidepressant drug imipramine (20 mg/kg body weight). Results Exposure to CUS for 28 days produced depression-like behavior in rats, as indicated by significant decreases in sucrose consumption, locomotor activity including decreased BDNF, Akt and CREB levels in the hippocampus. Daily administration of BME at a dose of (80 mg/kg body weight) significantly reverses the behavioral alteration and restored the normal level of BDNF, total and phospho-Akt, total and phospho CREB in the hippocampus of CUS induced rats as compared to vehicle treated control rats. Conclusion These findings suggest that BME ameliorates CUS induced behavioural depression in rats and that can be used as a potent therapeutic agent in treating depressive like behavior. PMID:28096878

  7. CREB regulation of BK channel gene expression underlies rapid drug tolerance

    PubMed Central

    Wang, Yan; Ghezzi, Alfredo; Yin, Jerry C.P.; Atkinson, Nigel S.

    2009-01-01

    Pharmacodynamic tolerance is believed to involve homeostatic mechanisms initiated to restore normal neural function. Drosophila exposed to a sedating dose of an organic solvent, such as benzyl alcohol or ethanol, acquire tolerance to subsequent sedation by that solvent. The slo gene encodes BK type Ca2+-activated K+ channels and has been linked to alcohol- and organic solvent-induced behavioral tolerance in mice, C. elegans and Drosophila. The cAMP response element binding (CREB) proteins are transcription factors that have been mechanistically linked to some behavioral changes associated with drug addiction. Here we show that benzyl alcohol sedation alters expression of both dCREB-A and dCREB2-b genes to increase production of positively acting CREB isoforms and to reduce expression of negatively acting CREB variants. Using a CREB-responsive reporter gene we show that benzyl alcohol sedation increases CREB-mediated transcription. Chromatin immunoprecipitation assays show that the binding of dCREB2, with a phosphorylated kinase-inducible domain, increases immediately after benzyl alcohol sedation within the slo promoter region. Most importantly, we show that a loss-of-function allele of dCREB2 eliminates drug-induced up-regulation of slo expression and the production of benzyl alcohol tolerance. This unambiguously links dCREB2 transcription factors to these two benzyl alcohol induced phenotypes. These findings suggest that CREB positively regulates the expression of slo-encoded BK type Ca2+-activated K+ channels, and that this gives rise to behavioral tolerance to benzyl alcohol sedation. PMID:19243452

  8. RGS17, an Overexpressed Gene in Human Lung and Prostate Cancer, Induces Tumor Cell Proliferation Through the Cyclic AMP-PKA-CREB Pathway

    PubMed Central

    James, Michael A.; Lu, Yan; Liu, Yan; Vikis, Haris G.; You, Ming

    2009-01-01

    We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth. PMID:19244110

  9. Silymarin suppresses the PGE2 -induced cell migration through inhibition of EP2 activation; G protein-dependent PKA-CREB and G protein-independent Src-STAT3 signal pathways.

    PubMed

    Woo, Seon Min; Min, Kyoung-Jin; Chae, In Gyeong; Chun, Kyung-Soo; Kwon, Taeg Kyu

    2015-03-01

    Silymarin has been known as a chemopreventive agent, and possesses multiple anti-cancer activities including induction of apoptosis, inhibition of proliferation and growth, and blockade of migration and invasion. However, whether silymarin could inhibit prostaglandin (PG) E2 -induced renal cell carcinoma (RCC) migration and what are the underlying mechanisms are not well elucidated. Here, we found that silymarin markedly inhibited PGE2 -stimulated migration. PGE2 induced G protein-dependent CREB phosphorylation via protein kinase A (PKA) signaling, and PKA inhibitor (H89) inhibited PGE2 -mediated migration. Silymarin reduced PGE2 -induced CREB phosphorylation and CRE-promoter activity. PGE2 also activated G protien-independent signaling pathways (Src and STAT3) and silymarin reduced PGE2 -induced phosphorylation of Src and STAT3. Inhibitor of Src (Saracatinib) markedly reduced PGE2 -mediated migration. We found that EP2, a PGE2 receptor, is involved in PGE2 -mediated cell migration. Down regulation of EP2 by EP2 siRNA and EP2 antagonist (AH6809) reduced PGE2 -inudced migration. In contrast, EP2 agonist (Butaprost) increased cell migration and silymarin effectively reduced butaprost-mediated cell migration. Moreover, PGE2 increased EP2 expression through activation of positive feedback mechanism, and PGE2 -induced EP2 expression, as well as basal EP2 levels, were reduced in silymarin-treated cells. Taken together, our study demonstrates that silymarin inhibited PGE2 -induced cell migration through inhibition of EP2 signaling pathways (G protein dependent PKA-CREB and G protein-independent Src-STAT3). © 2013 Wiley Periodicals, Inc.

  10. Differential regulation of c-jun and CREB by acrolein and 4-hydroxynonenal.

    PubMed

    Pugazhenthi, Subbiah; Phansalkar, Ketaki; Audesirk, Gerald; West, Anne; Cabell, Leigh

    2006-01-01

    In Alzheimer's disease (AD), oxidative stress-induced lipid peroxidation leads to accumulation of unsaturated aldehydes including acrolein and 4-hydroxynonenal (4HNE) in brain. In this study, we examined the effects of these lipid peroxidation products on apoptotic pathways in cultured neurons. Acrolein and 4HNE increased the levels of active phosphorylated forms of c-jun and CREB, the transcription factors that promote apoptosis and cell survival, respectively. However, they decreased the activity of CREB-dependent BDNF promoter while they increased the activity of promoters responsive to c-jun. We hypothesized that this differential regulation could be due to competition between proapoptotic c-jun and cytoprotective CREB for CBP (CREB-binding protein), a coactivator shared by several transcription factors. In support of this hypothesis, we demonstrate that the decrease of BDNF promoter activity by acrolein and 4HNE could be restored (i) by cotransfection with CBP, (ii) by cotransfection with VP 16-CREB, a constitutively active form of CREB that does not depend on CBP for its activation, or (iii) by inhibiting JNK-mediated c-jun activation. Finally, adenoviral transduction of hippocampal neurons with VP 16-CREB resulted in significant reduction in caspase-3 activation by acrolein and 4HNE. These observations suggest that lipid peroxidation-induced differential regulation of CREB and c-jun might play a role in neurodegeneration in AD.

  11. Essential role of CREB family proteins during Xenopus embryogenesis.

    PubMed

    Lutz, B; Schmid, W; Niehrs, C; Schütz, G

    1999-10-01

    The leucine zipper transcription factors cAMP response element binding protein (CREB), cAMP response element modulatory protein (CREM) and activating transcription factor 1 (ATF1) bind to the cAMP response element (CRE) with the palindromic consensus sequence TGACGTCA. Their transcriptional activities are dependent on serine phosphorylation induced by various extracellular signals such as hormones, growth factors and neurotransmitters. Here we show that CREB is the predominant CRE-binding protein in Xenopus embryos and that it plays an essential role during early development. The importance of CREB for morphogenetic processes was assessed by injection of RNA encoding a dominant-negative form of CREB that is fused to a truncated progesterone receptor ligand binding domain. In this fusion protein, a dominant-negative function can be induced by application of the synthetic steroid RU486 at given developmental stages. The inhibition of CREB at blastula and early gastrula stages leads to severe posterior defects of the embryos reflected by strong spina bifida, whereas the inhibition of CREB at the beginning of neurulation resulted in stunted embryos with microcephaly. In these embryos, initial induction of neural and mesodermal tissues is not dependent on CREB function, as genes such as Otx2, Krox20, Shh and MyoD are still expressed in injected embryos. But the expression domains of Otx2 and MyoD were found to be distorted reflecting the abnormal development in both neural and somitic derivatives. In summary, our data show that CREB is essential during several developmental stages of Xenopus embryogenesis.

  12. Illite improves memory impairment and reduces Aβ level in the Tg-APPswe/PS1dE9 mouse model of Alzheimer׳s disease through Akt/CREB and GSK-3β phosphorylation in the brain.

    PubMed

    Jeon, Songhee; Park, Jeong-Eun; Lee, Jinhee; Liu, Quan Feng; Jeong, Ha Jin; Pak, Sok Cheon; Yi, Sudok; Kim, Myung Hun; Kim, Chan-Wha; Park, Jung-Keug; Kim, Geun Woo; Koo, Byung-Soo

    2015-02-03

    The use of illite in Korean medicine has a long history as a therapeutic agent for various cerebrovascular diseases. According to Dongui Bogam, illite can be used for Qi-tonifying, phlegm dispersing and activation of blood circulation which is an important principle for the treatment of brain-associated diseases. This study was undertaken to evaluate beneficial effects of illite on the neurodegenerative diseases such as Alzheimer׳s disease (AD). The transgenic mice of AD, Tg-APPswe/PS1dE9, were fed with 1% or 3% of illite for 3 months. Behavioral, immunological and ELISA analyses were used to assess memory impairment with additional measurement of Aβ accumulation and plaque deposition in the brain. Other in vitro studies were performed to examine whether illite inhibits the Aβ-induced neurotoxicity in human neuroblastoma cell line, SH-SY5Y cells. Illite treatment rescued Aβ-induced neurotoxicity on SH-SY5Y cells, which was dependent on the PI3K/Akt activation. Intake of illite improved the Aβ-induced memory impairment and suppressed Aβ levels and plaque deposition in the brain of Tg-APPswe/PS1dE9 mice. Illite increased CREB, Akt, and GSK-3β phosphorylation and suppressed tau phosphorylation in the AD-like brains. Moreover, 1% of illite reduced weight gain and suppressed glucose level in the blood. The present study suggests that illite has the potential to be a useful adjunct as a therapeutic drug for the treatment of AD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Hippocampal cAMP/PKA/CREB is required for neuroprotective effect of acupuncture.

    PubMed

    Li, Qian-Qian; Shi, Guang-Xia; Yang, Jing-Wen; Li, Zhao-Xin; Zhang, Zhen-Hua; He, Tian; Wang, Jing; Liu, Li-Ying; Liu, Cun-Zhi

    2015-02-01

    Acupuncture has beneficial effects in vascular dementia (VaD) patients. The underlying mechanism, however, remains unknown. The present study was designed to investigate whether the cAMP/PKA/CREB cascade is involved in the mechanism of acupuncture in cerebral multi-infarction rats. In this study, cerebral multi-infarction was modeled in adult Wistar rats by homologous blood clot emboli. After a two-week acupuncture treatment at Zusanli (ST36), hippocampal-dependent memory was tested by employing a radial arm maze test. The hippocampus was isolated for analyses of cAMP concentration, phosphodiesterase (PDE) activity and CREB/pCREB and ERK/pERK expressions. The Morris water maze (MWM) task and CREB phosphorylation were evaluated in the presence of PKA-selective peptide inhibitor (H89). The radial arm maze test results demonstrated that acupuncture treatment at ST36 reversed hippocampal-dependent memory in impaired animals. Compared to those of the impaired group, cAMP concentration, PKA activity and pCREB and pERK expressions were increased following acupuncture therapy. Finally, the blockade of PKA reversed the increase in CREB phosphorylation and the improvement in recognitive function induced by acupuncture treatment. These results suggest that acupuncture could improve hippocampus function by modulating the cAMP/PKA/CREB signaling pathway, which represents a molecular mechanism of acupuncture for recognitive function in cerebral multi-infarction rats.

  14. MiR-582-5p/miR-590-5p targeted CREB1/CREB5–NF-κB signaling and caused opioid-induced immunosuppression in human monocytes

    PubMed Central

    Long, X; Li, Y; Qiu, S; Liu, J; He, L; Peng, Y

    2016-01-01

    Chronic opioid abusers are more susceptible to bacterial and viral infections, but the molecular mechanism underlying opioid-induced immunosuppression is unknown. MicroRNAs (miRNAs) are emerging as key players in the control of biological processes, and may participate in immune regulation. In this study, we investigated the molecular mechanisms in opioid-induced and miRNA-mediated immunosuppression, in the context of miRNA dysregulation in opioid abusers. Blood samples of heroin abusers were collected and analyzed using miRNA microarray analysis and quantitative PCR validation. The purified primary human monocytes were cultured in vitro to explore the underlying mechanism. We found that morphine and its derivative heroin significantly decreased the expression levels of miR-582-5p and miR-590-5p in monocytes. cAMP response element-binding protein 1 (CREB1) and CREB5 were detected as direct target genes of miR-582-5p and miR-590-5p, respectively, by using dual-luciferase assay and western bolt. Functional studies showed that knockdown of CREB1/CREB5 increased tumor necrosis factor alpha (TNF-α) level and enhanced expression of phospho–NF-κB p65 and NF-κB p65. Our results demonstrated that miR-582-5p and miR-590-5p play important roles in opioid-induced immunosuppression in monocytes by targeting CREB1/CREB5–NF-κB signaling pathway. PMID:26978739

  15. Involvement of PI3K/Akt/FoxO3a and PKA/CREB Signaling Pathways in the Protective Effect of Fluoxetine Against Corticosterone-Induced Cytotoxicity in PC12 Cells.

    PubMed

    Zeng, Bingqing; Li, Yiwen; Niu, Bo; Wang, Xinyi; Cheng, Yufang; Zhou, Zhongzhen; You, Tingting; Liu, Yonggang; Wang, Haitao; Xu, Jiangping

    2016-08-01

    The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.

  16. Neuronal Activity Rapidly Induces Transcription of the CREB-Regulated microRNA-132, in vivo

    PubMed Central

    Nudelman, Aaron S.; DiRocco, Derek P.; Lambert, Talley J.; Garelick, Michael G.; Le, Josh; Nathanson, Neil M.; Storm, Daniel R.

    2009-01-01

    Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 minutes and returning to basal levels within two hours of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity. PMID:19557767

  17. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity.

    PubMed

    Zhang, Juan; Tang, Hongju; Deng, Ruyuan; Wang, Ning; Zhang, Yuqing; Wang, Yao; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2015-01-01

    Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPβ expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPβ expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPβ was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPβ-triggered transcriptional cascades.

  18. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity

    PubMed Central

    Deng, Ruyuan; Wang, Ning; Zhang, Yuqing; Wang, Yao; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2015-01-01

    Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPβ expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPβ expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPβ was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPβ-triggered transcriptional cascades. PMID:25928058

  19. Tetramethylpyrazine protects against scopolamine-induced memory impairments in rats by reversing the cAMP/PKA/CREB pathway.

    PubMed

    Wu, Wei; Yu, Xiao; Luo, Xiao-Ping; Yang, Shu-Hua; Zheng, Dong

    2013-09-15

    Tetramethylpyrazine is used in the treatment of many neurological diseases because of its neuroprotective effect. Here, we demonstrate that administration of tetramethylpyrazine effectively reverses memory deficits induced by scopolamine. Moreover, tetramethylpyrazine preserves postsynaptic protein synthesis and restores cAMP/PKA/CREB pathway signaling deficits. Our study not only explores the actions of tetramethylpyrazine on synapses, but also provides novel evidence for the possible therapeutic use of tetramethylpyrazine in dementia.

  20. β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

    PubMed Central

    Cheng, Yun; Gao, Wei-Wei; Tang, Hei-Man Vincent; Deng, Jian-Jun; Wong, Chi-Ming; Chan, Chi-Ping; Jin, Dong-Yan

    2016-01-01

    CREB-H is an endoplasmic reticulum-resident bZIP transcription factor which critically regulates lipid homeostasis and gluconeogenesis in the liver. CREB-H is proteolytically activated by regulated intramembrane proteolysis to generate a C-terminally truncated form known as CREB-H-ΔTC, which translocates to the nucleus to activate target gene expression. CREB-H-ΔTC is a fast turnover protein but the mechanism governing its destruction was not well understood. In this study, we report on β-TrCP-dependent ubiquitination and proteasomal degradation of CREB-H-ΔTC. The degradation of CREB-H-ΔTC was mediated by lysine 48-linked polyubiquitination and could be inhibited by proteasome inhibitor. CREB-H-ΔTC physically interacted with β-TrCP, a substrate recognition subunit of the SCFβ-TrCP E3 ubiquitin ligase. Forced expression of β-TrCP increased the polyubiquitination and decreased the stability of CREB-H-ΔTC, whereas knockdown of β-TrCP had the opposite effect. An evolutionarily conserved sequence, SDSGIS, was identified in CREB-H-ΔTC, which functioned as the β-TrCP-binding motif. CREB-H-ΔTC lacking this motif was stabilized and resistant to β-TrCP-induced polyubiquitination. This motif was a phosphodegron and its phosphorylation was required for β-TrCP recognition. Furthermore, two inhibitory phosphorylation sites close to the phosphodegron were identified. Taken together, our work revealed a new intracellular signaling pathway that controls ubiquitination and degradation of the active form of CREB-H transcription factor. PMID:27029215

  1. Reduced phosphoCREB in Müller glia during retinal degeneration in rd10 mice

    PubMed Central

    Bachleda, Amelia; Xiong, Yubin; Osawa, Shoji; Weiss, Ellen R.

    2017-01-01

    Purpose The mechanisms that trigger retinal degeneration are not well understood, despite the availability of several animal models with different mutations. In the present report, the rd10 mouse, a model for retinitis pigmentosa (RP) that contains a mutation in the gene for PDE6β (Pde6b), is used to evaluate gliosis, as a marker for retinal stress, and cyclic AMP response element binding protein (CREB) phosphorylation, which may be important early in retinal degeneration. Methods Wild-type C57Bl6J and rd10 mice raised under cyclic light were examined for changes in gliosis and CREB phosphorylation for approximately 3 weeks beginning at P14 to P17 using immunocytochemistry. Mice raised under normal cyclic light and in complete darkness were also compared for changes in CREB phosphorylation. Results Gliosis in rd10 mice raised under cyclic light was apparent at P17, before extensive degeneration of the photoreceptor layer is visible, and increased over time. Phosphorylation of CREB at Ser133 (pCREB) was detected in Müller glia (MG) in the wild-type and rd10 mice. However, at all phases of photoreceptor degeneration, the pCREB levels were lower in the rd10 mice. We also observed extensive migration of MG cell bodies to the outer nuclear layer (ONL) during degeneration. In contrast to the mice raised under cyclic light, the rd10 mice raised in the dark exhibited slower rates of degeneration. When the dark-reared mice were exposed to cyclic light, the photoreceptor layer degenerated within 4 days to approximately one to two rows of nuclei. Interestingly, the pCREB levels in the MG also decreased during this 4-day cyclic light exposure compared to the levels in the rd10 mice raised continuously in the dark. Conclusions The results of these studies suggest that photoreceptors communicate directly or indirectly with MG at early stages, inducing gliosis before extensive retinal degeneration is apparent in rd10 mice. Surprisingly, phosphorylation of CREB is downregulated

  2. CD99 isoforms regulate CD1a expression in human monocyte-derived DCs through ATF-2/CREB-1 phosphorylation.

    PubMed

    Mahiddine, Karim; Mallavialle, Aude; Bziouech, Hanen; Larbret, Frédéric; Bernard, Alain; Bernard, Ghislaine

    2016-06-01

    CD1a expression is considered one of the major characteristics qualifying in vitro human dendritic cells (DCs) during their generation process. Here, we report that CD1A transcription is regulated by a mechanism involving the long and short isoforms of CD99. Using a lentiviral construct encoding for a CD99 short hairpin RNA, we were able to inhibit CD99 expression in human primary DCs. In such cells, CD1a membrane expression increased and CD1A transcripts were much higher in abundance compared to cells expressing CD99 long form (CD99LF). We also show that CD1A transcription is accompanied by a switch in expression from CD99LF to expression at comparable levels of both CD99 isoforms during immature DCs generation in vitro. We demonstrate that CD99LF maintains a lower level of CD1A transcription by up-regulating the phosphorylated form of the ATF-2 transcription factor and that CD99 short form (SF) is required to counteract this regulatory mechanism. Elucidation of the molecular mechanisms related to CD99 alternative splicing will be very helpful to better understand the transcriptional regulatory mechanism of CD1a molecules during DCs differentiation and its involvement in the immune response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    PubMed Central

    Almeida, Luis E. F.; Murray, Peter D.; Zielke, H. Ronald; Roby, Clinton D.; Kingsbury, Tami J.; Krueger, Bruce K.

    2009-01-01

    Cyclic AMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, CREB. However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of L-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Taken together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca2+ entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks. PMID:19812345

  4. Renal Proximal Tubule Na,K-ATPase is Controlled by CREB Regulated Transcriptional CoActivators as well as Salt Inducible Kinase 1

    PubMed Central

    Taub, Mary; Garamella, Sudha; Kim, Dongwook; Rajkhowa, Trivikram; Cutuli, Facundo

    2015-01-01

    Sodium reabsorption by the kidney is regulated by locally produced natriuretic and anti-natriuretic factors, including dopamine and norepinephrine, respectively. Previous studies indicated that signaling events initiated by these natriuretic and anti-natriuretic factors achieve their effects by altering the phosphorylation of Na,K-ATPase in the renal proximal tubule, and that Protein Kinase A (PKA) and Calcium mediated signaling pathways are involved. The same signaling pathways also control the transcription of the Na,K-ATPase β subunit gene atp1b1 in renal proximal tubule cells. In this report, evidence is presented that 1) both the recently discovered cAMP-Regulated Transcriptional Coactivators (CRTCs), and Salt Inducible Kinase 1 (SIK1) contribute to the transcriptional regulation of atp1b1 in renal proximal tubule (RPT) cells, and 2) that renal effectors including norepinephrine, dopamine, prostaglandins and sodium play a role. Exogenously expressed CRTCs stimulate atp1b1 transcription. Evidence for a role of endogenous CRTCs includes the loss of transcriptional regulation of atp1b1 by a dominant negative CRTC, as well as by a CREB mutant, with an altered CRTC binding site. In a number of experimental systems, SIK phosphorylates CRTCs, which are then sequestered in the cytoplasm, preventing their nuclear effects. Consistent with such a role of SIK in primary RPT cells, atp1b1 transcription increased in the presence of a dominant negative SIK1, and in addition, regulation by dopamine, norepinephrine and monensin was disrupted by a dominant negative SIK1. These latter observations can be explained, if SIK1 is phosphorylated and inactivated in the presence of these renal effectors. Our results support the hypothesis that Na,K-ATPase in the renal proximal tubule is regulated at the transcriptional level via SIK1 and CRTCs by renal effectors, in addition to the previously reported control of the phosphorylation of Na,K-ATPase. PMID:26432356

  5. Measuring CREB activation using bioluminescent probes that detect KID-KIX interaction in living cells.

    PubMed

    Ishimoto, Tetsuya; Mano, Hiroki; Ozawa, Takeaki; Mori, Hisashi

    2012-05-16

    The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300. The increase in the light intensity of the probe proteins results from the phosphorylation of the responsible serine corresponding to Ser-133 of CREB. Because these proteins have a high signal-to-noise ratio and are nontoxic, it has become possible for the first time to carry out long-term measurement of KID-KIX interaction in living cells. Furthermore, we examined the usefulness of the probe proteins for future high-throughput cell-based drug screening and found several herbal extracts that activated CREB.

  6. Stochasticity and bifurcations in a reduced model with interlinked positive and negative feedback loops of CREB1 and CREB2 stimulated by 5-HT.

    PubMed

    Hao, Lijie; Yang, Zhuoqin; Bi, Yuanhong

    2016-04-01

    The cyclic AMP (cAMP)-response element-binding protein (CREB) family of transcription factors is crucial in regulating gene expression required for long-term memory (LTM) formation. Upon exposure of sensory neurons to the neurotransmitter serotonin (5-HT), CREB1 is activated via activation of the protein kinase A (PKA) intracellular signaling pathways, and CREB2 as a transcriptional repressor is relieved possibly via phosphorylation of CREB2 by mitogen-activated protein kinase (MAPK). Song et al. [18] proposed a minimal model with only interlinked positive and negative feedback loops of transcriptional regulation by the activator CREB1 and the repressor CREB2. Without considering feedbacks between the CREB proteins, Pettigrew et al. [8] developed a computational model characterizing complex dynamics of biochemical pathways downstream of 5-HT receptors. In this work, to describe more simply the biochemical pathways and gene regulation underlying 5-HT-induced LTM, we add the important extracellular sensitizing stimulus 5-HT as well as the product Ap-uch into the Song's minimal model. We also strive to examine dynamical properties of the gene regulatory network under the changing concentration of the stimulus, [5-HT], cooperating with the varying positive feedback strength in inducing a high state of CREB1 for the establishment of long-term memory. Different dynamics including monostability, bistability and multistability due to coexistence of stable steady states and oscillations is investigated by means of codimension-2 bifurcation analysis. At the different positive feedback strengths, comparative analysis of deterministic and stochastic dynamics reveals that codimension-1 bifurcation with respect to [5-HT] as the parameter can predict diverse stochastic behaviors resulted from the finite number of molecules, and the number of CREB1 molecules more and more preferentially resides near the high steady state with increasing [5-HT], which contributes to long

  7. cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

    PubMed Central

    Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2013-01-01

    Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

  8. The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

    PubMed Central

    Menga, Alessio; Iacobazzi, Vito; Infantino, Vittoria; Avantaggiati, Maria Laura; Palmieri, Ferdinando

    2015-01-01

    The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. PMID:25597433

  9. Hemorrhage induces rapid in vivo activation of CREB and NF-kappaB in murine intraparenchymal lung mononuclear cells.

    PubMed

    Shenkar, R; Abraham, E

    1997-02-01

    Increased expression of proinflammatory cytokines appears to be an important factor contributing to the development of acute lung injury. In murine models, mRNA levels of proinflammatory and immunoregulatory cytokines, including IL-1alpha, IL-1beta, TGF-beta1, and TNF-alpha, are increased in intraparenchymal lung mononuclear cells 1 h after hemorrhage. Binding elements for the nuclear transcriptional regulatory factors, nuclear factor kappaB (NF-kappaB), CCAAT/enhancer binding protein beta (C/EBPbeta), serum protein 1 (Sp1), activator protein 1 (AP-1), and the cyclic AMP response-element binding protein (CREB) are present in the promoter regions of numerous cytokine genes, including those whose expression is increased after blood loss. To investigate early transcriptional mechanisms which may be involved in regulating pulmonary cytokine expression after hemorrhage, we examined in vivo activation of these five nuclear transcriptional factors among intraparenchymal lung mononuclear cells obtained in the immediate post-hemorrhage period. Activation of NF-kappaB and CREB, but not C/EBPbeta, Sp1, or AP-1, was present in lung mononuclear cells isolated from mice 15 min after hemorrhage. Inhibition of xanthine oxidase by prior feeding with either an allopurinol-supplemented or a tungsten-enriched diet prevented hemorrhage-induced activation of CREB, but not NF-kappaB. These results demonstrate that hemorrhage leads to rapid in vivo activation in the lung of CREB through a xanthine oxidase-dependent mechanism and of NF-kappaB through other pathways, and suggest that the activation of these transcriptional factors may have an important role in regulating pulmonary cytokine expression and the development of acute lung injury after blood loss.

  10. Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

    PubMed Central

    Liu, Fu-Chin; Graybiel, Ann M.

    1998-01-01

    The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine. PMID:9539803

  11. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    PubMed

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants.

  12. Changes in CREB and deltaFosB are associated with the behavioural sensitization induced by methylenedioxypyrovalerone.

    PubMed

    Buenrostro-Jáuregui, Mario; Ciudad-Roberts, Andres; Moreno, Josep; Muñoz-Villegas, Patricia; López-Arnau, Raúl; Pubill, David; Escubedo, Elena; Camarasa, Jorge

    2016-07-01

    Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone which has recently emerged as a designer drug of abuse. The objective of this study was to investigate the locomotor sensitization induced by MDPV in adolescent mice, and associated neuroplastic changes in the nucleus accumbens and striatum through deltaFosB and CREB expression. Behavioural testing consisted of three phases: Phase I: conditioning regimen with MDPV (0.3 mg/kg/day for five days) or saline; Phase II: resting (11 days); Phase III: challenged with MDPV (0.3 mg/kg), cocaine (10 mg/kg) or saline on day 16 for both groups. Mice repeatedly exposed to MDPV increased locomotor activity by 165-200% following acute MDPV or cocaine administration after an 11-day resting period, showing a MDPV-induced sensitization to itself and to cocaine. An explanation for this phenomenon could be the common mechanism of action between these two psychostimulants. Furthermore, the MDPV challenge resulted in higher levels of phospho-CREB in MDPV-conditioned mice compared with MDPV-naive mice, probably due to an up-regulation of the cAMP pathway. Likewise, MDPV exposure induced a persistent increase in the striatal expression of deltaFosB; the priming dose of MDPV also produced a significant increase in the accumbal expression of this transcription factor. This study constitutes the first evidence that an exposure to a low dose of MDPV during adolescence induces behavioural sensitization and provides a neurobiological basis for a relationship between MDPV and cocaine. We hypothesize that, similar to cocaine, both CREB and deltaFosB play a role in the induction of this behavioural sensitization. © The Author(s) 2016.

  13. CREB-mediated synaptogenesis and neurogenesis is crucial for the role of 5-HT1a receptors in modulating anxiety behaviors.

    PubMed

    Zhang, Jing; Cai, Cheng-Yun; Wu, Hai-Yin; Zhu, Li-Juan; Luo, Chun-Xia; Zhu, Dong-Ya

    2016-07-12

    Serotonin 1a-receptor (5-HT1aR) has been specifically implicated in the pathogenesis of anxiety. However, the mechanism underlying the role of 5-HT1aR in anxiety remains poorly understood. Here we show in mice that the transcription factor cAMP response element binding protein (CREB) in the hippocampus functions as an effector of 5-HT1aR in modulating anxiety-related behaviors. We generated recombinant lentivirus LV-CREB133-GFP expressing a dominant negative CREB which could not be phosphorylated at Ser133 to specifically reduce CREB activity, and LV-VP16-CREB-GFP expressing a constitutively active fusion protein VP16-CREB which could be phosphorylated by itself to specifically enhance CREB activity. LV-CREB133-GFP neutralized 5-HT1aR agonist-induced up-regulation of synapse density, spine density, dendrite complexity, neurogenesis, and the expression of synapsin and spinophilin, two well-characterized synaptic proteins, and abolished the anxiolytic effect of 5-HT1aR agonist; whereas LV-VP16-CREB-GFP rescued the 5-HT1aR antagonist-induced down-regulation of synapse density, spine density, dendrite complexity, neurogenesis and synapsin and spinophilin expression, and reversed the anxiogenic effect of 5-HT1aR antagonist. The deletion of neurogenesis by irradiation or the diminution of synaptogenesis by knockdown of synapsin expression abolished the anxiolytic effects of both CREB and 5-HT1aR activation. These findings suggest that CREB-mediated hippoacampus structural plasticity is crucial for the role of 5-HT1aR in modulating anxiety-related behaviors.

  14. CREB-mediated synaptogenesis and neurogenesis is crucial for the role of 5-HT1a receptors in modulating anxiety behaviors

    PubMed Central

    Zhang, Jing; Cai, Cheng-Yun; Wu, Hai-Yin; Zhu, Li-Juan; Luo, Chun-Xia; Zhu, Dong-Ya

    2016-01-01

    Serotonin 1a-receptor (5-HT1aR) has been specifically implicated in the pathogenesis of anxiety. However, the mechanism underlying the role of 5-HT1aR in anxiety remains poorly understood. Here we show in mice that the transcription factor cAMP response element binding protein (CREB) in the hippocampus functions as an effector of 5-HT1aR in modulating anxiety-related behaviors. We generated recombinant lentivirus LV-CREB133-GFP expressing a dominant negative CREB which could not be phosphorylated at Ser133 to specifically reduce CREB activity, and LV-VP16-CREB-GFP expressing a constitutively active fusion protein VP16-CREB which could be phosphorylated by itself to specifically enhance CREB activity. LV-CREB133-GFP neutralized 5-HT1aR agonist-induced up-regulation of synapse density, spine density, dendrite complexity, neurogenesis, and the expression of synapsin and spinophilin, two well-characterized synaptic proteins, and abolished the anxiolytic effect of 5-HT1aR agonist; whereas LV-VP16-CREB-GFP rescued the 5-HT1aR antagonist-induced down-regulation of synapse density, spine density, dendrite complexity, neurogenesis and synapsin and spinophilin expression, and reversed the anxiogenic effect of 5-HT1aR antagonist. The deletion of neurogenesis by irradiation or the diminution of synaptogenesis by knockdown of synapsin expression abolished the anxiolytic effects of both CREB and 5-HT1aR activation. These findings suggest that CREB-mediated hippoacampus structural plasticity is crucial for the role of 5-HT1aR in modulating anxiety-related behaviors. PMID:27404655

  15. Cyclophilin D deficiency rescues Aβ-impaired PKA/CREB signaling and alleviates synaptic degeneration.

    PubMed

    Du, Heng; Guo, Lan; Wu, Xiaoping; Sosunov, Alexander A; McKhann, Guy M; Chen, John Xi; Yan, Shirley ShiDu

    2014-12-01

    The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aβ)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aβ-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aβ-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aβ-induced ROS. Consequently, CypD-deficient neurons are resistant to Aβ-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aβ-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway.

  16. Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

    PubMed

    Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

  17. CREB is activated by UVC through a p38/HOG-1-dependent protein kinase.

    PubMed Central

    Iordanov, M; Bender, K; Ade, T; Schmid, W; Sachsenmaier, C; Engel, K; Gaestel, M; Rahmsdorf, H J; Herrlich, P

    1997-01-01

    Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway. PMID:9118940

  18. Enriched environment prevents hypobaric hypoxia induced memory impairment and neurodegeneration: role of BDNF/PI3K/GSK3β pathway coupled with CREB activation.

    PubMed

    Jain, Vishal; Baitharu, Iswar; Prasad, Dipti; Ilavazhagan, Govindasamy

    2013-01-01

    Adverse environmental conditions such as hypobaric hypoxia (HH) cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE) is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF) plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders.

  19. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    SciTech Connect

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li . E-mail: lfang@utmb.edu; Li Junfa . E-mail: junfali@cpums.edu.cn

    2006-02-10

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning.

  20. Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

    PubMed Central

    Steven, André; Leisz, Sandra; Sychra, Katharina; Hiebl, Bernhard; Wickenhauser, Claudia; Mougiakakos, Dimitrios; Kiessling, Rolf; Denkert, Carsten; Seliger, Barbara

    2016-01-01

    The cAMP-responsive element-binding protein (CREB) is involved in the tumorigenicity of HER-2/neu-overexpressing murine and human tumor cells, but a link between the HER-2/neu-mediated CREB activation, its posttranslational modification and localization and changes in the cellular metabolism, due to an altered (tumor) microenvironment remains to be established. The present study demonstrated that shRNA-mediated silencing of CREB in HER-2/neu-transformed cells resulted in decreased tumor formation, which was associated with reduced angiogenesis, but increased necrotic and hypoxic areas in the tumor. Hypoxia induced pCREBSer133, but not pCREBSer121 expression in HER-2/neu-transformed cells. This was accompanied by upregulation of the hypoxia-inducible genes GLUT1 and VEGF, increased cell migration and matrix metalloproteinase-mediated invasion. Treatment of HER-2/neu+ cells with signal transduction inhibitors targeting in particular HER-2/neu was able to revert hypoxia-controlled CREB activation. In addition to changes in the phosphorylation, hypoxic response of HER-2/neu+ cells caused a transient ubiquitination and SUMOylation as well as a co-localization of nuclear CREB to the mitochondrial matrix. A mitochondrial localization of CREB was also demonstrated in hypoxic areas of HER-2/neu+ mammary carcinoma lesions. This was accompanied by an altered gene expression pattern, activity and metabolism of mitochondria leading to an increased respiratory rate, oxidative phosphorylation and mitochondrial membrane potential and consequently to an enhanced apoptosis and reduced cell viability. These data suggest that the HER-2/neu-mediated CREB activation caused by a hypoxic tumor microenvironment contributes to the neoplastic phenotype of HER-2/neu+ cells at various levels. PMID:27409833

  1. Estrogen receptor α L429 and A430 regulate 17β-estradiol-induced cell proliferation via CREB1.

    PubMed

    Pesiri, Valeria; Totta, Pierangela; Segatto, Marco; Bianchi, Fabrizio; Pallottini, Valentina; Marino, Maria; Acconcia, Filippo

    2015-12-01

    17β-Estradiol (E2)-dependent cell proliferation requires both estrogen receptor α (ERα)-based integrated control of gene transcription and kinase pathways activation. Such coordination of intracellular E2:ERα-dependent signaling mechanisms is finely tuned by receptor association with specific partner proteins. Recently, we identified the leucine (L) 429 and alanine (A) 430 within the ERα ligand binding domain as important residues for receptor non-covalent interaction to ubiquitinated species [i.e., ERα ubiquitin-binding surface (ERα UBS)] and for E2-induced ERα activation. To date, if these two ERα amino acids are involved in the control of E2-dependent pathways required for cell proliferation is unknown. Here, by using stably expressing ERα mutated in L429 and A430 (i.e., L429A,A430G-LAAG) cell lines, we show that L429 and A430 are critical for E2-induced cell proliferation, PI3K/AKT pathway activation, and ERα-mediated transcriptional changes. Moreover, we demonstrate that these two receptor structural determinants direct the E2-induced PI3K/AKT/CREB1 pathway activation and CREB1-mediated transcriptional activity that in turn control the hormone-induced cell proliferation. As a whole, our data demonstrate for the first time that the ERα UBS contributes to the modulation of E2-induced ERα-mediated cell proliferation and provide a novel connection between the receptor structure and the functional molecular mechanisms by which E2:ERα complex can regulate cell processes.

  2. CREB1/ATF1 Activation in Photoreceptor Degeneration and Protection

    PubMed Central

    Beltran, William A.; Allore, Heather G.; Johnson, Elizabeth; Towle, Virginia; Tao, Weng; Acland, Gregory M.; Aguirre, Gustavo D.

    2009-01-01

    Purpose. The cAMP response element binding protein 1 (CREB1) and activating transcription factor 1 (ATF1) are closely related members of the bZIP superfamily of transcription factors. Both are activated in response to a wide array of stimuli, including cellular stress. This study was conducted to assess the CREB1/ATF1 pathway in photoreceptor disease and protection. Methods. The expression levels of p-CREB1, CREB1, and ATF1 were examined by immunoblot and immunohistochemistry in normal canine retina and retinas of several canine models of retinal degeneration (rcd1, rcd2, erd, prcd, XLPRA1, XLPRA2, T4R RHO). Humans retinas affected with age-related macular degeneration (AMD) were also examined. p-CREB1/ATF1 immunolabeling was assessed in normal and rcd1 dogs treated with ciliary neurotrophic factor (CNTF), to examine the effect of a neuroprotective stimulus on activation of CREB1/ATF1. Results. Native CREB1 and ATF1 as well as phosphorylated CREB1/ATF1 was examined in normal canine retina by immunoblot. The p-CREB1 antibody identified phosphorylated CREB1 and ATF1 and labeled the inner retina only in normal dogs. In degenerate canine and human retinas, strong immunolabeling appeared in rod and cone photoreceptors, indicating increased expression of native CREB1 and ATF1, as well as increased phosphorylation of these proteins. Retinal protection by CNTF in rcd1 dogs was accompanied by a significant increase in the number of p-CREB1/ATF1-labeled photoreceptor nuclei. Conclusions. Positive association of CREB1/ATF1 phosphorylation with photoreceptor protection suggests that it may contribute to an innate protective response. These data identify a signaling mechanism in rods and cones of potential importance for therapies of RP and AMD. PMID:19643965

  3. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    PubMed

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Oxytocin is involved in the proconvulsant effects of Sildenafil: Possible role of CREB.

    PubMed

    Khoshneviszadeh, Mahsima; Rahimian, Reza; Fakhfouri, Gohar; Payandemehr, Borna; Khodagholi, Fariba; Ejtemaei Mehr, Shahram; Dehpour, Ahmad Reza

    2016-08-10

    Sildenafil is a phosphodiesterase type 5 inhibitor mainly used for male erectile dysfunction. One of rare yet serious adverse effects of Sildenafil is its potential to decrease seizure threshold. Ample evidence suggests that Sildenafil exerts central effects through induction of Oxytocin (OT) secretion and CREB phosphorylation. The aim of the present study is to evaluate potential roles of OT and CREB in the proconvulsant effects of Sildenafil. The Pentylenetetrazole-induced seizure was used as a standard convulsion model in this study. OT release and pCREB expression were evaluated in the hippocampus of mice using ELISA and western blot assays, respectively. Our results showed that Sildenafil at the dose of 10mgkg(-1) or higher, significantly decreased seizure threshold. Pretreatment with a non-effective dose of OT, potentiated while OT receptor antagonist, Atosiban, reversed fully the proconvulsant effects of Sildenafil (5mgkg(-1)). At biochemical inspection, Sildenafil markedly increased CREB which was attenuated by coadministration of Atosiban. The present study shows for the first time that OT release and the subsequent CREB phosphorylation are involved in the proconvulsant effects of acute Sildenafil treatment in an experimental model of seizure.

  5. Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

    NASA Technical Reports Server (NTRS)

    Degani, CH.; Kawatsuji, M.; Halmann, M.

    1975-01-01

    It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

  6. Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

    NASA Technical Reports Server (NTRS)

    Degani, CH.; Kawatsuji, M.; Halmann, M.

    1975-01-01

    It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

  7. Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites.

    PubMed Central

    Pillay, T S; Xiao, S; Olefsky, J M

    1996-01-01

    Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling. PMID:8609215

  8. Methamphetamine induces Shati/Nat8L expression in the mouse nucleus accumbens via CREB- and dopamine D1 receptor-dependent mechanism

    PubMed Central

    Uno, Kyosuke; Miyazaki, Toh; Sodeyama, Kengo; Miyamoto, Yoshiaki

    2017-01-01

    Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction. PMID:28319198

  9. The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.

    PubMed Central

    Kvietikova, I; Wenger, R H; Marti, H H; Gassmann, M

    1995-01-01

    The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription. Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF1 probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing. Images PMID:8524640

  10. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    SciTech Connect

    Li, Mei; Zhang, Dong-Qing; Wang, Xiang-Zhen; Xu, Tie-Jun

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.

  11. Systemic Inhibition of CREB is Well-tolerated in vivo

    PubMed Central

    Li, Bingbing X.; Gardner, Ryan; Xue, Changhui; Qian, David Z.; Xie, Fuchun; Thomas, George; Kazmierczak, Steven C.; Habecker, Beth A.; Xiao, Xiangshu

    2016-01-01

    cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics. PMID:27694829

  12. Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

    SciTech Connect

    Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena; Accordi, Benedetta; Basso, Giuseppe; Gaspar, John Antonydas; Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina; Sachinidis, Agapios; Bremer-Hoffmann, Susanne

    2014-10-15

    According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.

  13. Role of Phosphorylated HDAC4 in Stroke-Induced Angiogenesis

    PubMed Central

    Liu, Juan; Zhou, Xiang; Li, Qing; Zhou, Shu-Min; Hu, Bin; Hu, Guo-Wen; Niu, Xin; Guo, Shang-Chun

    2017-01-01

    Acetylation or deacetylation of chromatin proteins and transcription factors is part of a complex signaling system that is involved in the control of neurological disorders. Recent studies have demonstrated that histone deacetylases (HDACs) exert protective effects in attenuating neuronal injury after ischemic insults. Class IIa HDAC4 is highly expressed in the brain, and neuronal activity depends on the nucleocytoplasmic shuttling of HDAC4. However, little is known about HDAC4 and its roles in ischemic stroke. In this study, we report that phosphorylation of HDAC4 was remarkably upregulated after stroke and blockade of HDAC4 phosphorylation with GÖ6976 repressed stroke-induced angiogenesis. Phosphorylation of HDAC4 was also increased in endothelial cells hypoxia model and suppression of HDAC4 phosphorylation inhibited the tube formation and migration of endothelial cells in vitro. Furthermore, in addition to the inhibition of angiogenesis, blockade of HDAC4 phosphorylation suppressed the expression of genes downstream of HIF-VEGF signaling in vitro and in vivo. These data indicate that phosphorylated HDAC4 may serve as an important regulator in stroke-induced angiogenesis. The protective mechanism of phosphorylated HDAC4 is associated with HIF-VEGF signaling, implicating a novel therapeutic target in stroke. PMID:28127553

  14. Morphine-induced conditioned place preference and the alterations of p-ERK, p-CREB and c-fos levels in hypothalamus and hippocampus: the effects of physical stress.

    PubMed

    Pahlevani, P; Fatahi, Z; Moradi, M; Haghparast, A

    2014-12-08

    The hypothalamus and hippocampus are important areas involved in stress responses and reward processing. In addition, ERK/CREB pathway plays a critical role in the control of cellular responses to stress and reward. In the current study, effects of acute and subchronic stress on the alteration of p-ERK, p-CREB and c-fos levels in the hypothalamus and hippocampus of saline- or morphine-treated animals during morphine-induced conditioned place preference (CPP) procedure were investigated. Male Wistar rats were divided into two saline- and morphine-treated supergroups. Each supergroup includes of control, acute stress and subchronic stress groups. In all of groups, the CPP procedure was done, afterward the alternation of p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level in the hypothalamus and hippocampus were estimated by Western blot analysis. The results indicated that in saline- or morphine-treated animals, p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level increased after application of acute and subchronic stress (except for p-ERK/ERK ratio in morphine-control group). Our findings revealed that in saline- or morphine-treated animals, acute and subcronic stress increased the p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level in the hypothalamus and hippocampus and this enhancement in morphine-treated animals, was more considerable than that in saline-treated animals.

  15. Neuroprotective action of N-acetyl serotonin in oxidative stress-induced apoptosis through the activation of both TrkB/CREB/BDNF pathway and Akt/Nrf2/Antioxidant enzyme in neuronal cells.

    PubMed

    Yoo, Jae-Myung; Lee, Bo Dam; Sok, Dai-Eun; Ma, Jin Yuel; Kim, Mee Ree

    2017-04-01

    N-acetyl serotonin (NAS) as a melatonin precursor has neuroprotective actions. Nonetheless, it is not clarified how NAS protects neuronal cells against oxidative stress. Recently, we have reported that N-palmitoyl serotonins possessed properties of antioxidants and neuroprotection. Based on those, we hypothesized that NAS, a N-acyl serotonin, may have similar actions in oxidative stress-induced neuronal cells, and examined the effects of NAS based on in vitro and in vivo tests. NAS dose-dependently inhibited oxidative stress-induced cell death in HT-22 cells. Moreover, NAS suppressed glutamate-induced apoptosis by suppressing expression of AIF, Bax, calpain, cytochrome c and cleaved caspase-3, whereas it enhanced expression of Bcl-2. Additionally, NAS improved phosphorylation of tropomyosin-related kinase receptor B (TrkB) and cAMP response element-binding protein (CREB) as well as expression of brain-derived neurotrophic factor (BDNF), whereas the inclusion of each inhibitor of JNK, p38 or Akt neutralized the neuroprotective effect of NAS, but not that of ERK. Meanwhile, NAS dose-dependently reduced the level of reactive oxygen species, and enhanced the level of glutathione in glutamate-treated HT-22 cells. Moreover, NAS significantly increased expression of heme oxygenase-1, NAD(P)H quinine oxidoreductase-1 and glutamate-cysteine ligase catalytic subunit as well as nuclear translocation of NF-E2-related factor-2. Separately, NAS at 30mg/kg suppressed scopolamine-induced memory impairment and cell death in CA1 and CA3 regions in mice. In conclusion, NAS shows actions of antioxidant and anti-apoptosis by activating TrkB/CREB/BDNF pathway and expression of antioxidant enzymes in oxidative stress-induced neurotoxicity. Therefore, such effects of NAS may provide the information for the application of NAS against neurodegenerative diseases.

  16. Chronic light deprivation inhibits appetitive associative learning induced by ethanol and its respective c-Fos and pCREB expression.

    PubMed

    Varela, Patrícia; Escosteguy-Neto, João Carlos; Coelho, Carolina Tesone; Mello, Luiz Eugênio; da Silveira, Dartiu Xavier; Santos-Junior, Jair Guilherme

    2014-11-01

    To address the role of mixed anxiety/mood disorder on appetitive associative learning, we verify whether previous chronic light deprivation changes ethanol-induced conditioned place preference and its respective expression of c-Fos and pCREB, markers of neuronal activity and plasticity. The experimental group was maintained in light deprivation for 24 h for a period of 4 wk. Subsequently, it was adapted to a standard light-dark cycle for 1 wk. As a control, some mice were maintained in standard cycle for a period of 4 wk (Naïve group). Then, all animals were submitted to behavioral tests to assess emotionality: elevated plus maze; open field; and forced swim. After that, they were submitted to ethanol-induced conditioned place preference. Ninety minutes after the place preference test, they were perfused, and their brains processed for c-Fos and pCREB immunohistochemistry. Light deprivation induced anxiety-like trait (elevated plus maze), despair (forced swim), and hyperlocomotion (open field), common features seen in other animal models of depression. Ethanol-induced conditioned place preference was accompanied by increases on c-Fos and pCREB in the hippocampus, prefrontal cortex and striatum. Interestingly, mice previously submitted to light deprivation did not develop either acquisition and/or expression of ethanol-induced conditioned place preference or increases in c-Fos and pCREB. Therefore, chronic light deprivation mimics several behavioral aspects of other animal models of depression. Furthermore, it could be useful to study the neurochemical mechanisms involved in the dual diagnosis. However, given its likely deleterious effects on appetitive associative memory, it should be used with caution to investigate the cognitive aspects related to the dual diagnosis.

  17. Enhancement of behavioral sensitization, anxiety-like behavior, and hippocampal and frontal cortical CREB levels following cocaine abstinence in mice exposed to cocaine during adolescence.

    PubMed

    Valzachi, Maria Cristina; Teodorov, Elizabeth; Marcourakis, Tania; Bailey, Alexis; Camarini, Rosana

    2013-01-01

    Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system.

  18. Activation of CREB by St. John's wort may diminish deletorious effects of aging on spatial memory.

    PubMed

    Trofimiuk, Emil; Holownia, Adam; Braszko, Jan J

    2010-03-01

    St. John's wort (Hypericum perforatum) is one of the leading psychotherapeutic phytomedicines. Beneficial effects of this herb in the treatment of mild to moderate depression are well known. In this study we tested a hypothesis that St. John's wort alleviates age-related memory impairments by increasing the levels of cyclic adenosine 3', 5'-monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) in hippocampus. Middleaged rats (18 month-old) displayed a decline in the acquisition of spatial working memory (p < 0.001) in the Morris water maze (MWM). Chronic administration of Hypericum perforatum (HP) (350 mg/kg for 21 days), potently and significantly improved the processing of spatial information in the aged rats (p < 0.001). Also the herb increased the levels of pCREB in the aged rat's hippocampus (p < 0.01) as measured by western immunoblotting. Aging caused significant locomotor impairments as tested in the open field (p < 0.001) but not in the MWM test. However, these were unaffected by treatment with HP. Thus, this study indicates that St. John's wort effectively prevents aging-induced deterioration of spatial memory in 18 month-old rats, possibly by the activation of CREB regulated genes associated with memory formation. It appears that mechanism is probably inactive in young rats.

  19. Involvement of BDNF/TrkB and ERK/CREB axes in nitroglycerin-induced rat migraine and effects of estrogen on these signals in the migraine

    PubMed Central

    Guo, Jiu-Qing; Deng, Hui-Hui; Bo, Xiao

    2017-01-01

    ABSTRACT Migraine is a highly prevalent headache disorder, especially in women. Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinases (TrkB), as well as extracellular signal-regulated kinase (ERK) and its downstream target c-AMP-responsive element binding protein (CREB) are strongly associated with the transmission of nociceptive information. However, the involvement of these substances in migraine has rarely been examined. In the present study, intraperitoneal injection of nitroglycerin (NTC) successfully induced rat migraine attack, as evidenced by behavioral testing. The location and abundance of these substances in the migraine model were determined by immunohistochemistry, real-time polymerase chain reaction (RT-PCR), western blot and enzyme-linked immunosorbant assays (ELISA). Results showed that BDNF, TrkB, phosphor(p)-ERK and p-CREB were up-regulated in the brain neurons of both male and female rats with NTG-induced migraine compared to non-migraine control, whereas their expression levels were decreased in headache-free intervals of the migraine compared to migraine attacks. Estrogen is an important contributor to migraine. Female ovariectomized rats showed significant reduction in the expression of BDNF, TrkB, p-CREB and p-ERK in both attacks and intervals of NTG-induced migraine, relative to rats that have their ovaries. But, intraperitoneal administration of exogenous estrogen recovered their expression in ovariectomized rats. Collectively, this study unveiled a positive correlation of BDNF/TrkB and ERK/CREB axes in NTG-induced migraine and promoting effects of estrogen on their signals in the migraine. These findings contribute to further understanding the pathogenesis of migraine in the molecular basis. PMID:27875242

  20. Differential regulation of cyclooxygenase-2 and inducible nitric oxide synthase by 4-hydroxynonenal in human osteoarthritic chondrocytes through ATF-2/CREB-1 transactivation and concomitant inhibition of NF-kappaB signaling cascade.

    PubMed

    Vaillancourt, France; Morquette, Barbara; Shi, Qin; Fahmi, Hassan; Lavigne, Patrick; Di Battista, John A; Fernandes, Julio C; Benderdour, Mohamed

    2007-04-01

    4-hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues and was recently identified as a potent catabolic factor in OA cartilage. In this study, we provide additional evidence that HNE acts as an inflammatory mediator by elucidating the signaling cascades targeted in OA chondrocytes leading to cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression. HNE induced COX-2 protein and mRNA levels with accompanying increases in prostaglandin E2 (PGE(2)) production. In contrast, HNE had no effect on basal iNOS expression or nitric oxide (NO) release. However, HNE strongly inhibited IL-1beta-induced iNOS or NO production. Transient transfection experiments revealed that the ATF/CRE site (-58/-53) is essential for HNE-induced COX-2 promoter activation and indeed HNE induced ATF-2 and CREB-1 phosphorylation as well as ATF/CRE binding activity. Overexpression of p38 MAPK enhanced the HNE-induced ATF/CRE luciferase reporter plasmid activation, COX-2 synthesis and promoter activity. HNE abrogated IL-1beta-induced iNOS expression and promoter activity mainly through NF-kappaB site (-5,817/-5,808) possibly via suppression of IKKalpha-induced IkappaBalpha phosphorylation and NF-kappaB/p65 nuclear translocation. Upon examination of upstream signaling components, we found that IKKalpha was inactivated through HNE/IKKalpha adduct formation. Taken together, these findings illustrate the central role played by HNE in the regulation of COX-2 and iNOS in OA. The aldehyde induced selectively COX-2 expression via ATF/CRE activation and inhibited iNOS via IKKalpha inactivation. c 2006 Wiley-Liss, Inc.

  1. ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

    PubMed

    Leduc, Michele; Richard, Joy; Costes, Safia; Muller, Dany; Varrault, Annie; Compan, Vincent; Mathieu, Julia; Tanti, Jean-François; Pagès, Gilles; Pouyssegur, Jacques; Bertrand, Gyslaine; Dalle, Stéphane; Ravier, Magalie A

    2017-07-18

    Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 (-/-) mice) and pharmacological approaches. NAD(P)H, free cytosolic Ca(2+) concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 (-/-) mouse beta cells (p < 0.01) compared with Erk1 (+/+) mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 (-/-) mice was slightly increased in young animals (4-month-old mice) vs Erk1 (+/+) mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean

  2. CREB Overexpression Ameliorates Age-related Behavioral and Biophysical Deficits

    NASA Astrophysics Data System (ADS)

    Yu, Xiao-Wen

    Age-related cognitive deficits are observed in both humans and animals. Yet, the molecular mechanisms underlying these deficits are not yet fully elucidated. In aged animals, a decrease in intrinsic excitability of pyramidal neurons from the CA1 sub-region of hippocampus is believed to contribute to age-related cognitive impairments, but the molecular mechanism(s) that modulate both these factors has yet to be identified. Increasing activity of the transcription factor cAMP response element-binding protein (CREB) in young adult rodents has been shown to facilitate cognition, and increase intrinsic excitability of their neurons. However, how CREB changes with age, and how that impacts cognition in aged animals, is not clear. Therefore, we first systematically characterized age- and training-related changes in CREB levels in dorsal hippocampus. At a remote time point after undergoing behavioral training, levels of total CREB and activated CREB (phosphorylated at S133, pCREB) were measured in both young and aged rats. We found that pCREB, but not total CREB was significantly reduced in dorsal CA1 of aged rats. Importantly, levels of pCREB were found to be positively correlated with short-term spatial memory in both young and aged rats i.e. higher pCREB in dorsal CA1 was associated with better spatial memory. These findings indicate that an age-related deficit in CREB activity may contribute to the development of age-related cognitive deficits. However, it was still unclear if increasing CREB activity would be sufficient to ameliorate age-related cognitive, and biophysical deficits. To address this question, we virally overexpressed CREB in CA1, where we found the age-related deficit. Young and aged rats received control or CREB virus, and underwent water maze training. While control aged animals exhibited deficits in long-term spatial memory, aged animals with CREB overexpression performed at levels comparable to young animals. Concurrently, aged neurons

  3. Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

    PubMed

    Qian, Xu; Li, Xinjian; Cai, Qingsong; Zhang, Chuanbao; Yu, Qiujing; Jiang, Yuhui; Lee, Jong-Ho; Hawke, David; Wang, Yugang; Xia, Yan; Zheng, Yanhua; Jiang, Bing-Hua; Liu, David X; Jiang, Tao; Lu, Zhimin

    2017-03-02

    Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

  4. Varicella-Zoster Virus Activates CREB, and Inhibition of the pCREB-p300/CBP Interaction Inhibits Viral Replication In Vitro and Skin Pathogenesis In Vivo

    PubMed Central

    François, Sylvie; Sen, Nandini; Mitton, Bryan; Xiao, Xiangshu; Sakamoto, Kathleen M.

    2016-01-01

    ABSTRACT Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro. CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development. IMPORTANCE Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs

  5. The cyclic AMP response element-binding protein antisense oligonucleotide induced anti-nociception and decreased the expression of KIF17 in spinal cord after peripheral nerve injury in mice

    PubMed Central

    Bo, Jinhua; Zhang, Wei; Sun, Xiaofeng; Yang, Yan; Liu, Xiaojie; Jiang, Ming; Ma, Zhengliang; Gu, Xiaoping

    2014-01-01

    Backgrounds: The cyclic AMP response element-binding protein (CREB) plays an important role in neuropathic pain. Kinesin superfamily motor protein 17 (KIF17) is involved in long-term memory formation. CREB could increase the level of KIF17 when activated by synaptic input. This study is to investigate the role and mechanism of CREB antisense oligonucleotide (ODN) in neuropathic pain induced by chronic constriction injury (CCI) in mice. Results: CCI surgery decreased thresholds of mechanical allodynia and thermal hyperalgesia whereas CREB antisense oligonucleotide ODN significantly attenuated these pain behaviors (P < 0.05). CCI significantly induced the protein expression of phosphorylated CREB (pCREB) and KIF17, but not KIF5B, in the spinal cord of CCI mice (P < 0.05). Additionally, the mRNA expression of CREB and KIF17 was significantly increased by CCI (P < 0.05). However, CREB antisense ODN significantly decreased the protein expression of pCREB and KIF17 (but not KIF5B), and the mRNA expression of CREB and KIF17 (P < 0.05). Conclusions: CREB antisense oligonucleotide ODN may reduce neuropathic pain through targeting CREB and decreasing the expression of pCREB and KIF17. PMID:25664020

  6. Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways.

    PubMed

    Yan, Tingting; Zhao, Yan; Zhang, Xia; Lin, Xiaotong

    2016-03-10

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption.

  7. Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways

    PubMed Central

    Yan, Tingting; Zhao, Yan; Zhang, Xia; Lin, Xiaotong

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption. PMID:26978376

  8. CREB SUMOylation by the E3 ligase PIAS1 enhances spatial memory.

    PubMed

    Chen, Yan-Chu; Hsu, Wei-Lun; Ma, Yun-Li; Tai, Derek J C; Lee, Eminy H Y

    2014-07-16

    cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1Δ, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1Δ increased the expression level of CREB1Δ. CREB1Δ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumo-mutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

  9. Cranial irradiation regulates CREB-BDNF signaling and variant BDNF transcript levels in the mouse hippocampus.

    PubMed

    Son, Yeonghoon; Yang, Miyoung; Kang, Sohi; Lee, Sueun; Kim, Jinwook; Kim, Juhwan; Park, Seri; Kim, Joong-Sun; Jo, Sung-Kee; Jung, Uhee; Shin, Taekyun; Kim, Sung-Ho; Wang, Hongbing; Moon, Changjong

    2015-05-01

    The brain can be exposed to ionizing radiation in various ways, and such irradiation can trigger adverse effects, particularly on learning and memory. However, the precise mechanisms of cognitive impairments induced by cranial irradiation remain unknown. In the hippocampus, brain-derived neurotrophic factor (BDNF) plays roles in neurogenesis, neuronal survival, neuronal differentiation, and synaptic plasticity. The significance of BDNF transcript variants in these contexts is becoming clearer. In the present study, both object recognition memory and contextual fear conditioning task performance in adult C57BL/6 mice were assessed 1 month after a single exposure to cranial irradiation (10 Gy) to evaluate hippocampus-related behavioral dysfunction following such irradiation. Furthermore, changes in the levels of BDNF, the cAMP-response element binding protein (CREB) phosphorylation, and BDNF transcript variants were measured in the hippocampus 1 month after cranial irradiation. On object recognition memory and contextual fear conditioning tasks, mice evaluated 1 month after irradiation exhibited significant memory deficits compared to sham-irradiated controls, but no apparent change was evident in locomotor activity. Both phosphorylated CREB and BDNF protein levels were significantly downregulated after irradiation of the hippocampus. Moreover, the levels of mRNAs encoding common BDNF transcripts, and exons IIC, III, IV, VII, VIII, and IXA, were significantly downregulated after irradiation. The reductions in CREB phosphorylation and BDNF expression induced by differential regulation of BDNF hippocampal exon transcripts may be associated with the memory deficits evident in mice after cranial irradiation.

  10. Environmental enrichment protects against stress-induced anxiety: Role of glucocorticoid receptor, ERK, and CREB signaling in the basolateral amygdala.

    PubMed

    Novaes, Leonardo S; Dos Santos, Nilton Barreto; Batalhote, Rafaela F P; Malta, Marília Brinati; Camarini, Rosana; Scavone, Cristoforo; Munhoz, Carolina Demarchi

    2017-02-01

    Environmental enrichment (EE) is an experimental animal model that enhances an animal's opportunity to interact with sensory, motor, and social stimuli, compared to standard laboratory conditions. A prominent benefit of EE is the reduction of stress-induced anxiety. The relationship between stress and the onset of anxiety-like behavior has been widely investigated in experimental research, showing a clear correlation with structural changes in the hippocampus and basolateral amygdala (BLA). However, the mechanisms by which EE exerts its protective roles in stress and anxiety remain unclear, and it is not known whether EE reduces the effects of acute stress on animal behavior shortly following the cessation of stress. We found that EE can prevent the emergence of anxiety-like symptoms in rats measured immediately after acute restraint stress (1 h) and this effect is not due to changes in systemic release of corticosterone. Rather, we found that stress promotes a rapid increase in the nuclear translocation of glucocorticoid receptor (GR) in the BLA, an effect prevented by previous EE exposure. Furthermore, we observed a reduction of ERK (a MAPK protein) and CREB activity in the BLA promoted by both EE and acute stress. Finally, we found that EE decreases the expression of the immediate-early gene EGR-1 in the BLA, indicating a possible reduction of neuronal activity in this region. Hyperactivity of BLA neurons has been reported to accompany anxiety-like behavior and changes in this process may be one of the mechanism by which EE exerts its protective effects against stress-induced anxiety. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Stress induces altered CRE/CREB pathway activity and BDNF expression in the hippocampus of glucocorticoid receptor-impaired mice.

    PubMed

    Alboni, Silvia; Tascedda, Fabio; Corsini, Daniela; Benatti, Cristina; Caggia, Federica; Capone, Giacomo; Barden, Nicholas; Blom, Joan M C; Brunello, Nicoletta

    2011-06-01

    The gene coding for the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) is a stress-responsive gene. Changes in its expression may underlie some of the pathological effects of stress-related disorders like depression. Data on the stress-induced regulation of the expression of BDNF in pathological conditions are rare because often research is conducted using healthy animals. In our experiments, we used transgenic mice with glucocorticoid receptor impaired (GR-i) expression in the hypothalamus created as a tool to study the neuroendocrine changes occurring in stress-related disorders. First, under basal condition, GR-i mice displayed lower levels of BDNF exons IX and IV and decreased CRE(BDNF) binding activity with respect to wild-type (WT) mice in the hippocampus. Then, we exposed GR-i and WT mice to an acute restraint stress (ARS) to test the hypothesis that GR-i mice display: 1] different ARS induced expression of BDNF, and 2] altered activation of signaling pathways implicated in regulating BDNF gene expression in the hippocampus with respect to WT mice. Results indicate that ARS enhanced BDNF mRNA expression mainly in the CA3 hippocampal sub-region of GR-i mice in the presence of enhanced levels of pro-BDNF protein, while no effect was observed in WT mice. Moreover, ARS reduced CREB signaling and binding to the BDNF promoter in GR-i mice but enhanced signaling and binding, possibly through ERK1/2 activation, in WT mice. Thus, life-long central GR dysfunction resulted in an altered sensitivity at the transcriptional level that may underlie an impaired response to an acute psycho-physical stress. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

  12. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

    PubMed

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P; Kunz, Stefan

    2013-05-01

    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

  13. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan

    PubMed Central

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P.; Kunz, Stefan

    2013-01-01

    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a hemorrhagic fever with high mortality in man. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process. PMID:23279385

  14. Endogenous prostaglandin E2 potentiates anti-inflammatory phenotype of macrophage through the CREB-C/EBP-β cascade.

    PubMed

    Na, Yi Rang; Jung, Daun; Yoon, Bo Ruem; Lee, Won Woo; Seok, Seung Hyeok

    2015-09-01

    Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-β expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-β promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-β cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-β expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 .

  15. Human T-Cell Lymphotropic/Leukemia Virus Type 1 Tax Abrogates p53-Induced Cell Cycle Arrest and Apoptosis through Its CREB/ATF Functional Domain

    PubMed Central

    Mulloy, J. C.; Kislyakova, T.; Cereseto, A.; Casareto, L.; LoMonico, A.; Fullen, J.; Lorenzi, M. V.; Cara, A.; Nicot, C.; Giam, C.-Z.; Franchini, G.

    1998-01-01

    Human T-cell lymphotropic/leukemia virus type 1 (HTLV-1) transforms human T cells in vitro, and Tax, a potent transactivator of viral and cellular genes, plays a key role in cell immortalization. Tax activity is mediated by interaction with cellular transcription factors including members of the CREB/ATF family, the NF-κB/c-Rel family, serum response factor, and the coactivators CREB binding protein-p300. Although p53 is usually not mutated in HTLV-1-infected T cells, its half-life is increased and its function is impaired. Here we report that transient coexpression of p53 and Tax results in the suppression of p53 transcriptional activity. Expression of Tax abrogates p53-induced G1 arrest in the Calu-6 cell line and prevents the apoptosis induced by overexpressing p53 in the HeLa/Tat cell line. The Tax mutants M22 and G148V, which selectively activate the CREB/ATF pathway, exert these same biological effects on p53 function. In contrast, the NF-κB-active Tax mutant M47 has no effect on p53 activity in any of these systems. Consistent with the negative effect of Tax on p53, no activity on a p53-responsive promoter was observed upon transfection of HTLV-1-infected T-cell lines. The p53 protein is expressed at high levels in the nucleus, and nuclear extracts of HTLV-1-infected T cells bind constitutively to a DNA oligonucleotide containing the p53 response element, indicating that Tax does not interfere with p53 binding to DNA. Tax is able to suppress the transactivation function of p53 in three different cell lines, and this suppression required Tax-mediated activation of the CREB/ATF, but not the NF-κB/c-Rel, pathway. Tax and the active Tax mutants were able to abrogate the G1 arrest and apoptosis induced by p53, and this effect does not correlate with an altered localization of nuclear p53 or with the disruption of p53-DNA complexes. The suppression of p53 activity by Tax could be important in T-cell immortalization induced by HTLV-1. PMID:9765430

  16. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    PubMed

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

    2015-01-01

    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

  17. Baicalin Attenuates Ketamine-Induced Neurotoxicity in the Developing Rats: Involvement of PI3K/Akt and CREB/BDNF/Bcl-2 Pathways.

    PubMed

    Zuo, Daiying; Lin, Li; Liu, Yumiao; Wang, Chengna; Xu, Jingwen; Sun, Feng; Li, Lin; Li, Zengqiang; Wu, Yingliang

    2016-08-01

    Ketamine is widely used as an anesthetic in pediatric clinical practice. However, numerous studies have reported that exposure to ketamine during the developmental period induces neurotoxicity. Here we investigate the neuroprotective effects of baicalin, a natural flavonoid compound, against ketamine-induced apoptotic neurotoxicity in the cortex and hippocampus of the Sprague-Dawley postnatal day 7 (PND7) rat pups. Our results revealed that five continuous injections of ketamine (20 mg/kg) at 90-min intervals over 6 h induced obvious morphological damages of neuron by Nissl staining and apoptosis by TUNEL assays in the prefrontal cortex and hippocampus of PND7 rat pups. Baicalin (100 mg/kg) pretreatment alleviated ketamine-induced morphological change and apoptosis. Caspase-3 activity and caspase-3 mRNA expression increase induced by ketamine were also inhibited by baicalin treatment. LY294002, an inhibitor of PI3K, abrogated the effect of baicalin against ketamine-induced caspase-3 activity and caspase-3 mRNA expression increase. In addition, Western blot studies indicated that baicalin not only inhibited ketamine-induced p-Akt and p-GSK-3β decrease, but also relieved ketamine-induced p-CREB and BDNF expression decrease. Baicalin also attenuated ketamine-induced Bcl-2/Bax decrease and caspase-3 expression increase. Further in vitro experiments proved that baicalin mitigated ketamine-induced cell viability decrease in the MTT assay, morphological change by Rosenfeld's staining, and caspase-3 expression increase by Western blot in the primary neuron-glia mixed cultures. LY294002 abrogated the protective effect of baicalin. These data demonstrate that baicalin exerts neuroprotective effect against ketamine-induced neuronal apoptosis by activating the PI3K/Akt and its downstream CREB/BDNF/Bcl-2 signaling pathways. Therefore, baicalin appears to be a promising agent in preventing or reversing ketamine's apoptotic neurotoxicity at an early developmental stage.

  18. Neuroprotective effects of various doses of topiramate against methylphenidate-induced oxidative stress and inflammation in isolated rat amygdala: the possible role of CREB/BDNF signaling pathway.

    PubMed

    Motaghinejad, Majid; Motevalian, Manijeh; Falak, Reza; Heidari, Mansour; Sharzad, Mahshid; Kalantari, Elham

    2016-12-01

    Methylphenidate (MPH) abuse damages brain cells. The neuroprotective effects of topiramate (TPM) have been reported previously, but its exact mechanism of action still remains unclear. This study investigated the in vivo role of various doses of TPM in the protection of rat amygdala cells against methylphenidate-induced oxidative stress and inflammation. Seventy adult male rats were divided into seven groups. Groups 1 and 2 received normal saline (0.7 ml/rat) and MPH (10 mg/kg), respectively, for 21 days. Groups 3, 4, 5, 6, and 7 were concurrently treated with MPH (10 mg/kg) and TPM (10, 30, 50, 70, and 100 mg/kg), respectively, for 21 days. elevated plus maze (EPM) was used to assess motor activity disturbances. In addition, oxidative, antioxidantand inflammatory factors and CREB, Ak1, CAMK4, MAPK3, PKA, BDNF, and c FOS gene levels were measured by RT-PCR, and also, CREB and BDNF protein levels were measured by WB in isolated amygdalae. MPH significantly disturbed motor activity and TPM (70 and 100 mg/kg) neutralized its effects. MPH significantly increased lipid peroxidation, mitochondrial GSSG levels and IL-1β and TNF-α level and CAMK4 gene expression in isolated amygdala cells. In contrast, superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and CREB, BDNF Ak1, MAPK3, PKA, BDNF, and c FOS expression significantly decreased. The various doses of TPM attenuated these effects of MPH. It seems that TPM can be used as a neuroprotective agent and is a good candidate against MPH-induced neurodegeneration.

  19. cAMP/PKA/CREB/GLT1 signaling involved in the antidepressant-like effects of phosphodiesterase 4D inhibitor (GEBR-7b) in rats

    PubMed Central

    Liu, Xu; Guo, Haibiao; Sayed, Mohammad Daud SOM; Lu, Yang; Yang, Ting; Zhou, Dongsheng; Chen, Zhongming; Wang, Haitao; Wang, Chuang; Xu, Jiangping

    2016-01-01

    Objectives GEBR-7b, a potential phosphodiesterase 4D inhibitor, has been shown to have memory-enhancing effects in rodents. However, it is still unknown whether GEBR-7b also has the antidepressant-like effects in rats. Herein, we examined the potential of GEBR-7b to attenuate depression-like behaviors in the rat model of depression induced by chronic unpredictable stress (CUS). Next, we also investigated the alterations of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) catalytic subunit (PKAca), cAMP response element-binding (CREB), and glutamate transporter 1 (GLT1) levels produced by GEBR-7b in the rats model of depression. Methods Effects of GEBR-7b on CUS (35 days)-induced depression-like behaviors were examined by measuring immobility time in the forced swimming test (FST). Hippocampal cAMP levels were examined by enzyme-linked immunosorbent assay, whereas PKAca, phosphorylation of CREB (pCREB), CREB, and GLT1 in the hippocampus of rats were subjected to Western blot analysis. Results CUS exposure caused a depression-like behavior evidenced by the increased immobility time in FST. Depression-like behavior induced by CUS was accompanied by a significant increased GLT, decreased cAMP, PKAca, pCREB activities in hippocampus. However, repeated GEBR-7b administration significantly reversed CUS-induced depression-like behavior and changes of cAMP/PKA/CREB/GLT1 signaling. No alteration was observed in locomotor activity in open field test. Conclusion These findings indicate that GEBR-7b reversed the depression-like behaviors induced by CUS in rats, which is at least in part mediated by modulating cAMP, PKAca, pCREB, and GLT1 levels in the hippocampus of rats, supporting its neuroprotective potential against behavioral and biochemical dysfunctions induced by CUS. PMID:26855578

  20. The Insulin-Regulated CREB Coactivator TORC Promotes Stress Resistance in Drosophila

    PubMed Central

    Wang, Biao; Goode, Jason; Best, Jennifer; Meltzer, Jodi; Schilman, Pablo E.; Chen, Jian; Garza, Dan; Thomas, John B.; Montminy, Marc

    2013-01-01

    SUMMARY In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance. PMID:18460334

  1. GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

    PubMed

    Zibrova, Darya; Vandermoere, Franck; Göransson, Olga; Peggie, Mark; Mariño, Karina V; Knierim, Anne; Spengler, Katrin; Weigert, Cora; Viollet, Benoit; Morrice, Nicholas A; Sakamoto, Kei; Heller, Regine

    2017-03-07

    Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

  2. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

    PubMed

    Ahluwalia, Amrita; Baatar, Dolgor; Jones, Michael K; Tarnawski, Andrzej S

    2014-09-15

    Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

  3. Cigarette sidestream smoke induces phosphorylated histone H2AX.

    PubMed

    Toyooka, Tatsushi; Ibuki, Yuko

    2009-05-31

    Cigarette sidestream smoke (CSS) is a widespread environmental pollutant having highly genotoxic potency. In spite of the overwhelming evidence that CSS induces a wide range of DNA damage such as oxidative base damage and DNA adducts, evidence that CSS can result in DNA double strand breaks (DSBs) is little. In this study, we showed that CSS generated phosphorylated histone H2AX (gamma-H2AX), recently considered as a sensitive marker of the generation of DSBs, in a human pulmonary epithelial cell model, A549. Treatment with CSS drastically induced discrete foci of gamma-H2AX within the nucleus in a dose-dependent manner. CSS increased intracellular oxidation, and N-acetylcysteine (NAC), an antioxidant, significantly attenuated the formation of gamma-H2AX, suggesting that reactive oxygen species produced from CSS partially contributed to the phosphorylation. The generation of gamma-H2AX is considered to be accompanied the induction of DSBs. CSS in fact induced DSBs, which was also inhibited by NAC. DSBs are the worst type of DNA damage, related to genomic instability and carcinogenesis. Our results would increase the evidence of the strong genotoxicity of passive smoking.

  4. AGE-RELATED IMPAIRMENTS IN MEMORY AND IN CREB AND pCREB EXPRESSION IN HIPPOCAMPUS AND AMYGDALA FOLLOWING INHIBITORY AVOIDANCE TRAINING

    PubMed Central

    Morris, Ken A.; Gold, Paul E.

    2012-01-01

    This experiment examined whether age-related changes in CREB and pCREB contribute to the rapid forgetting seen in aged animals. Young (3-month-old) and aged (24-month-old) Fischer-344 rats received inhibitory avoidance training with a low (0.2 mA, 0.4 sec) or moderate (0.5 mA, 0.5 sec) footshock; memory was measured 7 days later. Other rats were euthanized 30 minutes after training, and CREB and pCREB expression levels were examined in the hippocampus, amygdala, and piriform cortex using immunohistochemistry. CREB levels decreased with age in the hippocampus and amygdala. After training with either shock level, young rats exhibited good memory and increases in pCREB levels in the hippocampus and amygdala. Aged rats exhibited good memory for the moderate but not the low shock but did not show increases in pCREB levels after either shock intensity. These results suggest that decreases in total CREB and in pCREB activation in the hippocampus and amygdala may contribute to rapid forgetting in aged rats. After moderate footshock, the stable memory in old rats together with absence of CREB activation suggests either that CREB was phosphorylated in a spatiotemporal pattern other than analyzed here or that the stronger training conditions engaged alternate mechanisms that promote long-lasting memory. PMID:22445851

  5. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    NASA Technical Reports Server (NTRS)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

  6. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    NASA Technical Reports Server (NTRS)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

  7. Antiproliferative effect of panaxynol on RASMCs via inhibition of ERK1/2 and CREB.

    PubMed

    Jiang, Li-Ping; Lu, Yang; Nie, Bao-Ming; Chen, Hong-Zhuan

    2008-02-15

    Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.

  8. Phosphorylation induces distinct alpha-synuclein strain formation

    PubMed Central

    Ma, Meng-Rong; Hu, Zhi-Wen; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

    2016-01-01

    Synucleinopathies are a group of neurodegenerative diseases associated with alpha-synuclein (α-Syn) aggregation. Recently, increasing evidence has demonstrated the existence of different structural characteristics or ‘strains’ of α-Syn, supporting the concept that synucleinopathies share several common features with prion diseases and possibly explaining how a single protein results in different clinical phenotypes within synucleinopathies. In earlier studies, the different strains were generated through the regulation of solution conditions, temperature, or repetitive seeded fibrillization in vitro. Here, we synthesize homogeneous α-Syn phosphorylated at serine 129 (pS129 α-Syn), which is highly associated with the pathological changes, and demonstrate that phosphorylation at Ser129 induces α-Syn to form a distinct strain with different structures, propagation properties, and higher cytotoxicity compared with the wild-type α-Syn. The results are the first demonstration that post-translational modification of α-Syn can induce different strain formation, offering a new mechanism for strain formation. PMID:27853185

  9. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  10. Protective Effect of Curcumin by Modulating BDNF/DARPP32/CREB in Arsenic-Induced Alterations in Dopaminergic Signaling in Rat Corpus Striatum.

    PubMed

    Srivastava, Pranay; Dhuriya, Yogesh K; Gupta, Richa; Shukla, Rajendra K; Yadav, Rajesh S; Dwivedi, Hari N; Pant, Aditya B; Khanna, Vinay K

    2016-12-13

    Earlier, protective role of curcumin in arsenic-induced dopamine (DA)-D2 receptor dysfunctions in corpus striatum has been demonstrated by us. In continuation to that, the present study is focused to decipher the molecular mechanisms associated with alterations in dopaminergic signaling on arsenic exposure in corpus striatum and assess the protective efficacy of curcumin. Exposure to arsenic (20 mg/kg, body weight p.o. for 28 days) in rats resulted to decrease the expression of presynaptic proteins-tyrosine hydroxylase and VMAT2 while no effect was observed on the expression of DAT in comparison to controls. A significant decrease in the expression of DA-D2 receptors associated with alterations in the expression of PKA, pDARPP32 (Thr 34), and PP1 α was clearly evident on arsenic exposure. Expression of BDNF and pGSK3β in corpus striatum was found decreased in arsenic-exposed rats. Simultaneous treatment with curcumin (100 mg/kg, body weight p.o. for 28 days) resulted to protect arsenic-induced alterations in the expression of DA-D2 receptors, PKA, pDARPP32, pCREB, and pPP1α. Neuroprotective efficacy of curcumin can possibly be attributed to its antioxidant potential which significantly protected arsenic-induced mitochondrial dysfunctions by modulating the ROS generation and apoptosis. Modulation in the expression of BDNF and pGSK3β in corpus striatum by curcumin exhibits the importance of neuronal survival pathway in arsenic-induced dopaminergic dysfunctions. Interestingly, curcumin was also found to protect arsenic-induced ultrastructural changes in corpus striatum. The results exhibit that curcumin modulates BDNF/DARPP32/CREB in arsenic-induced alterations in dopaminergic signaling in rat corpus striatum.

  11. Thr175-phosphorylated tau induces pathologic fibril formation via GSK3β-mediated phosphorylation of Thr231 in vitro.

    PubMed

    Moszczynski, Alexander J; Gohar, May; Volkening, Kathryn; Leystra-Lantz, Cheryl; Strong, Wendy; Strong, Michael J

    2015-03-01

    We have previously shown that amyotrophic lateral sclerosis with cognitive impairment can be characterized by pathologic inclusions of microtubule-associated protein tau (tau) phosphorylated at Thr(175) (pThr(175)) in association with GSK3β activation. We have now examined whether pThr(175) induces GSK3β activation and whether this leads to pathologic fibril formation through Thr(231) phosphorylation. Seventy-two hours after transfection of Neuro2A cells with pseudophosphorylated green fluorescent protein-tagged 2N4R tau (Thr(175)Asp), phosphorylated kinase glycogen synthase kinase 3 beta (active GSK3β) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3β inhibitors or small hairpin RNA knockdown of GSK3β abolished fibril formation and prevented cell death. Inhibition of Thr(231) phosphorylation (Thr(231)Ala) prevented pathologic tau fibril formation, regardless of Thr(175) state, whereas Thr(231)Asp (pseudophosphorylated at Thr(231)) developed pathologic tau fibrils. Ser(235) mutations did not affect fibril formation, indicating an unprimed mechanism of Thr(231) phosphorylation. These findings suggest a mechanism of tau pathology by which pThr(175) induces GSK3β phosphorylation of Thr(231) leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB.

    PubMed

    Zhang, Yanling; Zhen, Wei; Maechler, Pierre; Liu, Dongmin

    2013-04-01

    Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of type 2 diabetes (T2D). Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of antiapoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and pancreatic and duodenal homeobox-1 (PDX-1) expression. Chronic hyperlipidemia significantly diminished cyclic adenosine monophosphate (cAMP) production, protein kinase A (PKA) activation, cAMP-responsive element binding protein (CREB) phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol-stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade.

  13. The selective glucocorticoid receptor antagonist ORG 34116 decreases immobility time in the forced swim test and affects cAMP-responsive element-binding protein phosphorylation in rat brain.

    PubMed

    Bachmann, Cornelius G; Bilang-Bleuel, Alicia; De Carli, Sonja; Linthorst, Astrid C E; Reul, Johannes M H M

    2005-01-01

    Glucocorticoid receptor (GR) antagonists can block the retention of the immobility response in the forced swimming test. Recently, we showed that forced swimming evokes a distinct spatiotemporal pattern of cAMP-responsive element-binding protein (CREB) phosphorylation in the dentate gyrus (DG) and neocortex. In the present study, we found that chronic treatment of rats with the selective GR antagonist ORG 34116 decreased the immobility time in the forced swim test, increased baseline levels of phosphorylated CREB (P-CREB) in the DG and neocortex and affected the forced swimming-induced changes in P-CREB levels in a time- and site-specific manner. Overall, we observed that, in control rats, forced swimming evoked increases in P-CREB levels in the DG and neocortex, whereas in ORG 34116-treated animals a major dephosphorylation of P-CREB was observed. These observations underscore an important role of GRs in the control of the phosphorylation state of CREB which seems to be of significance for the immobility response in the forced swim test and extend the molecular mechanism of action of GRs in the brain.

  14. Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

    ERIC Educational Resources Information Center

    Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

    2012-01-01

    Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

  15. Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

    ERIC Educational Resources Information Center

    Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

    2012-01-01

    Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

  16. Allicin inhibits oxidative stress-induced mitochondrial dysfunction and apoptosis by promoting PI3K/AKT and CREB/ERK signaling in osteoblast cells.

    PubMed

    Ding, Guoliang; Zhao, Jianquan; Jiang, Dianming

    2016-06-01

    Osteoporosis is a disease of the skeleton that is characterized by the loss of bone mass and degeneration of bone microstructure, resulting in an increased risk of fracture. Oxidative stress, which is known to promote oxidative damage to mitochondrial function and also cell apoptosis, has been recently indicated to be implicated in osteoporosis. However, there are few agents that counteract oxidative stress in osteoporosis. In the present study, the protective effects of allicin against the oxidative stress-induced mitochondrial dysfunction and apoptosis were investigated in murine osteoblast-like MC3T3-E1 cells. The results demonstrated that allicin counteracted the reduction of cell viability and induction of apoptosis caused by hydrogen peroxide (H2O2) exposure. The inhibition of apoptosis by allicin was confirmed by the inhibition of H2O2-induced cytochrome c release and caspase-3 activation. Moreover, the inhibition of apoptosis by allicin was identified to be associated with the counteraction of H2O2-induced mitochondrial dysfunction. In addition, allicin was demonstrated to be able to significantly ameliorate the repressed phosphoinositide 3-kinase (PI3K)/AKT and cyclic adenosine monophosphate response element-binding protein (CREB)/extracellular-signal-regulated kinase (ERK) signaling pathways by H2O2, which may also be associated with the anti-oxidative stress effects of allicin. In conclusion, allicin protects osteoblasts from H2O2-induced oxidative stress and apoptosis in MC3T3-E1 cells by improving mitochondrial function and the activation of PI3K/AKT and CREB/ERK signaling. The present study implies a promising role of allicin in oxidative stress-associated osteoporosis.

  17. Allicin inhibits oxidative stress-induced mitochondrial dysfunction and apoptosis by promoting PI3K/AKT and CREB/ERK signaling in osteoblast cells

    PubMed Central

    DING, GUOLIANG; ZHAO, JIANQUAN; JIANG, DIANMING

    2016-01-01

    Osteoporosis is a disease of the skeleton that is characterized by the loss of bone mass and degeneration of bone microstructure, resulting in an increased risk of fracture. Oxidative stress, which is known to promote oxidative damage to mitochondrial function and also cell apoptosis, has been recently indicated to be implicated in osteoporosis. However, there are few agents that counteract oxidative stress in osteoporosis. In the present study, the protective effects of allicin against the oxidative stress-induced mitochondrial dysfunction and apoptosis were investigated in murine osteoblast-like MC3T3-E1 cells. The results demonstrated that allicin counteracted the reduction of cell viability and induction of apoptosis caused by hydrogen peroxide (H2O2) exposure. The inhibition of apoptosis by allicin was confirmed by the inhibition of H2O2-induced cytochrome c release and caspase-3 activation. Moreover, the inhibition of apoptosis by allicin was identified to be associated with the counteraction of H2O2-induced mitochondrial dysfunction. In addition, allicin was demonstrated to be able to significantly ameliorate the repressed phosphoinositide 3-kinase (PI3K)/AKT and cyclic adenosine monophosphate response element-binding protein (CREB)/extracellular-signal-regulated kinase (ERK) signaling pathways by H2O2, which may also be associated with the anti-oxidative stress effects of allicin. In conclusion, allicin protects osteoblasts from H2O2-induced oxidative stress and apoptosis in MC3T3-E1 cells by improving mitochondrial function and the activation of PI3K/AKT and CREB/ERK signaling. The present study implies a promising role of allicin in oxidative stress-associated osteoporosis. PMID:27284348

  18. Neuregulin1 signaling targets SRF and CREB and activates the muscle spindle-specific gene Egr3 through a composite SRF-CREB-binding site.

    PubMed

    Herndon, Carter A; Ankenbruck, Nick; Lester, Bridget; Bailey, Julie; Fromm, Larry

    2013-03-10

    Muscle spindles are sensory receptors embedded within muscle that detect changes in muscle length. Each spindle is composed of specialized muscle fibers, known as intrafusal muscle fibers, along with the endings of axons from sensory neurons that innervate these muscle fibers. Formation of muscle spindles requires neuregulin1 (NRG1), which is released by sensory axons, activating ErbB receptors in muscle cells that are contacted. In muscle cells, the transcription factor Egr3 is transcriptionally induced by NRG1, which in turn activates various target genes involved in forming the intrafusal fibers of muscle spindles. The signaling relay within the NRG1-ErbB pathway that acts to induce Egr3 is presumably critical for muscle spindle formation but for the most part has not been determined. In the current studies, we examined, using cultured muscle cells, transcriptional regulatory mechanisms by which Egr3 responds to NRG1. We identified a composite regulatory element for the Egr3 gene, consisting adjacent sites that bind cAMP response element binding protein (CREB) and serum response factor (SRF), with a role in NRG1 responsiveness. The SRF element also influences Egr3 basal expression in unstimulated myotubes, and in the absence of the SRF element, the CREB element influences basal expression. We show that NRG1 signaling, to target SRF, acts on the SRF coactivators myocardian-related transcription factor (MRTF)-A and MRTF-B, which are known to activate SRF-mediated transcription, by stimulating their translocation from the cytoplasm to the nucleus. CREB is phosphorylated, which is known to contribute to its activation, in response to NRG1. These results suggest that NRG1 induces expression of the muscle spindle-specific gene Egr3 by stimulating the transcriptional activity of CREB and SRF.

  19. Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism

    PubMed Central

    Kim, Sang Hwa; Trinh, Anthony T.; Larsen, Michele Campaigne; Mastrocola, Adam S.; Jefcoate, Colin R.; Bushel, Pierre R.; Tibbetts, Randal S.

    2016-01-01

    cAMP response element binding protein (CREB) is a key regulator of glucose metabolism and synaptic plasticity that is canonically regulated through recruitment of transcriptional coactivators. Here we show that phosphorylation of CREB on a conserved cluster of Ser residues (the ATM/CK cluster) by the DNA damage-activated protein kinase ataxia-telangiectasia-mutated (ATM) and casein kinase1 (CK1) and casein kinase2 (CK2) positively and negatively regulates CREB-mediated transcription in a signal dependent manner. In response to genotoxic stress, phosphorylation of the ATM/CK cluster inhibited CREB-mediated gene expression, DNA binding activity and chromatin occupancy proportional to the number of modified Ser residues. Paradoxically, substoichiometric, ATM-independent, phosphorylation of the ATM/CK cluster potentiated bursts in CREB-mediated transcription by promoting recruitment of the CREB coactivator, cAMP-regulated transcriptional coactivators (CRTC2). Livers from mice expressing a non-phosphorylatable CREB allele failed to attenuate gluconeogenic genes in response to DNA damage or fully activate the same genes in response to glucagon. We propose that phosphorylation-dependent regulation of DNA binding activity evolved as a tunable mechanism to control CREB transcriptional output and promote metabolic homeostasis in response to rapidly changing environmental conditions. PMID:27431323

  20. Ethanol-induced phosphorylation of cytokeratin in cultured hepatocytes

    SciTech Connect

    Kawahara, Hiromu; Cadrin, M.; French, S.W. )

    1990-01-01

    The authors studied the effect of ethanol on the phosphorylation of cytokeratins (CKs) in cultured hepatocytes since CK filaments are resulted by phosphorylation and they are abnormal in alcoholic liver disease. Hepatocytes were obtained from 14-day-old rats and cultured for 48 hrs. The hepatocytes were exposed to ethanol for 30 min. The residual insoluble cytoskeletons were analyzed by two-dimensional gel electrophoresis and autoradiography. 2D gel electrophoresis showed CK 55 and CK 49 or 8 and 18 and actin. The CKs had several isoelectric variants. The most basic spot was the dominant protein which was not phosphorylated. The more acidic spots were phosphorylated. After ethanol treatment, the phosphorylation of CK 55 and CK 49 were markedly increased over controls. They compared these results, with the effect of vasopressin, TPA and db-cAMP on the phosphorylation of CKs. Vasopressin and TPA caused the phosphorylation of CK 55 and 49 but db-cAMP did not.

  1. 5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

    PubMed Central

    Giono, L E; Varone, C L; Cánepa, E T

    2001-01-01

    The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression. PMID:11139395

  2. Effect of rhynchophylline on the expression of p-CREB and sc-Fos in triatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference rats.

    PubMed

    Liu, Wei; Peng, Qiu-Xian; Lin, Xiao-Liang; Luo, Chao-Hua; Jiang, Ming-Jin; Mo, Zhi-Xian; Yung, Ken Kin-Lam

    2014-01-01

    To explore the effect of rhynchophylline (Rhy) on the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area of methamphetamine-induced conditioned place preference (CPP) rat, methamphetamine (2 mg/kg) was injected to rats and the conditioned place preference was observed in these rats treated with or without Rhy. An immunohistochemistry assay was used to determine the expression of p-CREB and c-Fos in the striatum and hippocampal CA1 area. Methamphetamine induced significant behavior alteration in CPP, while after pretreatment with rhynchophylline or ketamine, the time of staying in methamphetamine-paired compartment of rats was significantly reduced. Methamphetamine also increased the number of p-CREB positive cells in the striatum and hippocampal CA1 zone, as well as p-Fos positive cells. However, the compound Rhy could attenuate the effect. These findings show that Rhy can suppress the acquisition of CPP in rats induced by methamphetamine and the action may be related with the reduced expression of p-CREB and p-Fos in the striatum and hippocampus.

  3. Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

    PubMed Central

    Baik, Jiseok; Ok, Seong-Ho; Cho, Hyunhoo; Yu, Jongsun; Kim, Woochan; Nam, In-Koo; Choi, Mun-Jeoung; Lee, Heon-Keun; Sohn, Ju-Tae

    2014-01-01

    Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH2-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC. PMID:25332685

  4. Differences in basal and morphine-induced FosB/DeltaFosB and pCREB immunoreactivities in dopaminergic brain regions of alcohol-preferring AA and alcohol-avoiding ANA rats.

    PubMed

    Kaste, Kristiina; Kivinummi, Tanja; Piepponen, T Petteri; Kiianmaa, Kalervo; Ahtee, Liisa

    2009-06-01

    Besides alcohol, alcohol-preferring AA and alcohol-avoiding ANA rats differ also with respect to other abused drugs. To study the molecular basis of these differences, we examined the expression of two transcription factors implicated in addiction, DeltaFosB and pCREB, in brain dopaminergic regions of AA and ANA rats. The effects of morphine and nicotine were studied to relate the behavioral and molecular changes induced by these drugs. Baseline FosB/DeltaFosB immunoreactivity (IR) in the nucleus accumbens core and pCREB IR in the prefrontal cortex (PFC) were elevated in AA rats. Morphine increased DeltaFosB-like IR more readily in the caudate-putamen of AA rats than in ANA rats. In the PFC morphine decreased pCREB IR in AA rats, but increased it in ANA rats. In addition to enhanced locomotor response, the development of place preference to morphine was enhanced in AA rats. The enhanced nicotine-induced locomotor sensitization found in AA compared with ANA rats seems to depend in addition to dopamine and DeltaFosB on other mechanisms. These findings suggest that enhanced sensitivity of AA rats to morphine is related to augmented morphine-induced expression of FosB/DeltaFosB and morphine-induced reduction of pCREB levels. Moreover, altered innate expression of FosB/DeltaFosB and pCREB in AA rats is likely to affect the sensitivity of these rats to abused drugs.

  5. Synergistic Neuroprotective Effects of Two Herbal Ingredients via CREB-Dependent Pathway

    PubMed Central

    Liu, Xu; Wang, Dongxiao; Zhao, Runqing; Dong, Xianzhe; Hu, Yuan; Liu, Ping

    2016-01-01

    As two natural oligosaccharide esters, 3,6’-Disinapoyl sucrose (DISS) and tenuifolisideA (TFSA) are originating from the root of Polygala tenuifolia Willd, a traditional Chinese medicine used in treatment of mental disorders. Previous reports have shown that both of them possess in vitro neuroprotective effects by stimulating different upstream pathways related with cyclic AMP-responsive element-binding protein (CREB). In the present study, we investigated the additive neuroprotective effects of DISS and TFSA on Glu-induced damage of SY5Y cells and purposed the possible underlying mechanism. The interaction between DISS and TFSA showed a clear-cut synergistic effect as evidenced by combination index (CI). Additional evidence from biochemical (NOS activity) assays confirmed their additive inhibition on the Glu-induced NOS hyperactivation. Moreover, we showed that co-treatment of DISS and TFSA resulted in an additively up-regulated phosphorylation of CREB as well as increased expressions of CRTC1 and BDNF. Neuroprotective effects of DISS and TFSA on Glu-induced decrease in cell viability were blocked by MAPK/ERK1/2 inhibitor (U0126) and PI3-K inhibitor (LY290042). Nevertheless, the CRTC1 or BDNF expression induced by these two compounds was significantly reduced in the presence of either ERK or PI3-K inhibitor, indicating that the two oligosaccharide esters shared some common pathways in the regulation of CREB-BDNF pathway. Taken together, we, for the first time, showed that DISS and TFSA exerted the additive neuroprotective effects on CREB-BDNF signaling pathway through complementary mechanisms. PMID:27729863

  6. Synergistic Neuroprotective Effects of Two Herbal Ingredients via CREB-Dependent Pathway.

    PubMed

    Liu, Xu; Wang, Dongxiao; Zhao, Runqing; Dong, Xianzhe; Hu, Yuan; Liu, Ping

    2016-01-01

    As two natural oligosaccharide esters, 3,6'-Disinapoyl sucrose (DISS) and tenuifolisideA (TFSA) are originating from the root of Polygala tenuifolia Willd, a traditional Chinese medicine used in treatment of mental disorders. Previous reports have shown that both of them possess in vitro neuroprotective effects by stimulating different upstream pathways related with cyclic AMP-responsive element-binding protein (CREB). In the present study, we investigated the additive neuroprotective effects of DISS and TFSA on Glu-induced damage of SY5Y cells and purposed the possible underlying mechanism. The interaction between DISS and TFSA showed a clear-cut synergistic effect as evidenced by combination index (CI). Additional evidence from biochemical (NOS activity) assays confirmed their additive inhibition on the Glu-induced NOS hyperactivation. Moreover, we showed that co-treatment of DISS and TFSA resulted in an additively up-regulated phosphorylation of CREB as well as increased expressions of CRTC1 and BDNF. Neuroprotective effects of DISS and TFSA on Glu-induced decrease in cell viability were blocked by MAPK/ERK1/2 inhibitor (U0126) and PI3-K inhibitor (LY290042). Nevertheless, the CRTC1 or BDNF expression induced by these two compounds was significantly reduced in the presence of either ERK or PI3-K inhibitor, indicating that the two oligosaccharide esters shared some common pathways in the regulation of CREB-BDNF pathway. Taken together, we, for the first time, showed that DISS and TFSA exerted the additive neuroprotective effects on CREB-BDNF signaling pathway through complementary mechanisms.

  7. Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    PubMed Central

    Malaguarnera, Roberta; Sacco, Antonella; Morcavallo, Alaide; Squatrito, Sebastiano; Migliaccio, Antimo; Morrione, Andrea; Maggiolini, Marcello

    2014-01-01

    We have previously demonstrated that, in prostate cancer cells, androgens up-regulate IGF-I receptor (IGF-IR) by inducing cAMP-response element-binding protein (CREB) activation and CREB-dependent IGF-IR gene transcription through androgen receptor (AR)-dependent membrane-initiated effects. This IGF-IR up-regulation is not blocked by classical antiandrogens and sensitizes cells to IGF-I-induced biological effects. Metformin exerts complex antitumoral functions in various models and may inhibit CREB activation in hepatocytes. We, therefore, evaluated whether metformin may affect androgen-dependent IGF-IR up-regulation. In the AR+ LNCaP prostate cancer cells, we found that metformin inhibits androgen-induced CRE activity and IGF-IR gene transcription. CRE activity requires the formation of a CREB-CREB binding protein-CREB regulated transcription coactivator 2 (CRTC2) complex, which follows Ser133-CREB phosphorylation. Metformin inhibited Ser133-CREB phosphorylation and induced nuclear exclusion of CREB cofactor CRTC2, thus dissociating the CREB-CREB binding protein-CRTC2 complex and blocking its transcriptional activity. Similarly to metformin action, CRTC2 silencing inhibited IGF-IR promoter activity. Moreover, metformin blocked membrane-initiated signals of AR to the mammalian target of rapamycin/p70S6Kinase pathway by inhibiting AR phosphorylation and its association with c-Src. AMPK signals were also involved to some extent. By inhibiting androgen-dependent IGF-IR up-regulation, metformin reduced IGF-I-mediated proliferation of LNCaP cells. These results indicate that, in prostate cancer cells, metformin inhibits IGF-I-mediated biological effects by disrupting membrane-initiated AR action responsible for IGF-IR up-regulation and suggest that metformin could represent a useful adjunct to the classical antiandrogen therapy. PMID:24437490

  8. Acupuncture Activates ERK-CREB Pathway in Rats Exposed to Chronic Unpredictable Mild Stress

    PubMed Central

    Lu, Jun; Liang, Jia; Wang, Jun-Ren; Hu, Li; Tu, Ya; Guo, Jian-You

    2013-01-01

    Extracellular signal-regulated kinase (ERK)-cAMP response element binding protein (CREB) signal pathway has been implicated in the pathogenesis of depression. There is growing evidence that acupuncture in traditional Chinese medicine has antidepressant-like effect. However, the effect of acupuncture on ERK-CREB pathway remains unknown. In our study, the antidepressant-like effect of acupuncture treatment was measured by sucrose intake test and open field test in rats exposed to chronic unpredictable mild stress (CUMS) for 4 weeks. The protein levels of ERK1/2, CREB, phosphorylated ERK1/2 (p-ERK1/2), and phosphorylated CREB (p-CREB) in the hippocampus (HP) and prefrontal cortex (PFC) were examined by Western blot analysis. Our results showed that CUMS rats exhibited the reduction in behavioral activities, whereas acupuncture stimulation at acupoints Baihui (Du20) and Neiguan (PC6) reversed the behavioral deficit. In addition, exposure to CUMS resulted in the decrease of p-ERK1/2 and p-CREB in the HP and PFC. Acupuncture increased the ratio of p-ERK1/2 to ERK1/2 and the ratio of p-CREB to CREB in the HP and PFC. Our study suggested that one potential way, by which acupuncture had antidepressant-like effect, might be mediated by activating the ERK-CREB pathway in the brain. PMID:23843874

  9. Mdivi-1 Protects Against Ischemic Brain Injury via Elevating Extracellular Adenosine in a cAMP/CREB-CD39-Dependent Manner.

    PubMed

    Cui, Mei; Ding, Hongyan; Chen, Fangzhe; Zhao, Yanxin; Yang, Qi; Dong, Qiang

    2016-01-01

    This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

  10. Storage of passive motion pattern in hippocampal CA1 region depends on CaMKII/CREB signaling pathway in a motion sickness rodent model

    PubMed Central

    Wang, Junqin; Liu, Jiluo; Pan, Leilei; Qi, Ruirui; Liu, Peng; Zhou, Wei; Cai, Yiling

    2017-01-01

    Sensory mismatch between actual motion information and anticipated sensory patterns (internal model) is the etiology of motion sickness (MS). Some evidence supports that hippocampus might involve the neural storage of the “internal model”. This study established an “internal model” acquisition-retention behavioral model using a repeated habituation rotation training protocol. We tried to identify the hippocampal subregion involved in “internal model” retention using chemical lesion methods. Hippocampal kinases (CaMK, CaMKIV, CREB and ERK1/2) phosphorylation in the target subregion was assayed and the effects of kinase inhibitors (KN93 or U0126) on “internal model” retention were investigated. The activities of potential kinases (CaMKII and CREB) were also examined in otoliths deficit het/het mice. In habituated rats, CA1 lesion reproduced MS-related behavioral responses on “internal model” retention day. Habituation training increased CaMKII and CREB activity but had no effect on CaMKIV and ERK1/2 activity in the CA1, while inhibition of CaMKII but not ERK1/2 impaired “internal model” retention. In het/het mice, CaMKII and CREB were not activated in the CA1 on the retention day. These results suggested that CaMKII/CREB pathway might potentially contribute to the storage of the “internal model” in the hippocampal CA1 after motion sickness induced by vestibular stimulation. PMID:28230177

  11. Essential oil of Syzygium aromaticum reverses the deficits of stress-induced behaviors and hippocampal p-ERK/p-CREB/brain-derived neurotrophic factor expression.

    PubMed

    Liu, Bin-Bin; Luo, Liu; Liu, Xiao-Long; Geng, Di; Li, Cheng-Fu; Chen, Shao-Mei; Chen, Xue-Mei; Yi, Li-Tao; Liu, Qing

    2015-02-01

    Syzygium aromaticum has been widely used in traditional medicine. Our study investigated the safety and antidepressant-like effects of the essential oil of S. aromaticum after acute or long-term treatment. Using GC-MS, a total of eight volatile constituents were identified in the essential oil of S. aromaticum. The single LD50 was approximately 4500 mg/kg based on a 24-h acute oral toxicity study. In a long-term repeated toxicity study of this essential oil (100, 200, and 400 mg/kg, p. o.), only 400 mg/kg induced a significant decrease in body weight. In addition, no significant changes in relative organ weights and histopathological analysis were observed in all doses of essential oil-treated mice compared with the control group. Furthermore, acute S. aromaticum essential oil administration by gavage exerted antidepressant-like effects in the forced swimming test (200 mg/kg, p < 0.05) and tail suspension test (100 and 200 mg/kg, p < 0.05). Long-term S. aromaticum essential oil treatment via gavage significantly increased sucrose preference (50 mg/kg, p < 0.05; 100 and 200 mg/kg, p < 0.01) as well as elevated the protein levels of hippocampal p-ERK, p-CREB, and brain-derived neurotrophic factor in mice exposed to chronic unpredictable mild stress. These results confirmed the safety of the essential oil of S. aromaticum and suggested that its potent antidepressant-like property might be attributed to the improvement in the hippocampal pERK1/2-pCREB-BDNF pathway in rats exposed to chronic unpredictable mild stress. Georg Thieme Verlag KG Stuttgart · New York.

  12. Expression Patterns of CREBs in Oocyte Growth and Maturation of Fish

    PubMed Central

    Wang, De-Shou; Sudhakumari, Cheni-Chery; Kobayashi, Tohru; Nagahama, Yoshitaka

    2015-01-01

    In fish, oocyte meiotic maturation is regulated by 17α, 20β-dihydroxy-progesterone through cAMP. To study the role of cAMP response element binding protein (CREB) in meiotic maturation, we cloned and characterized the expression pattern of CREBs from two fish models, the Nile tilapia and catfish. In the Nile tilapia three different CREBs were identified where in CREB1 was found in many tissues including gonads with abundant expression in testis. CREB2, few amino acids shorter than CREB1, was expressed in several tissues with abundant expression in ovary. In addition, a 3’UTR variant form, CREB3 was exclusively found in ovary. During natural 14-day ovarian cycle of the Nile tilapia, CREB1 expression was stable throughout vitellogenesis with a sharp decrease on the day of spawning. In contrast, CREB2 remain unchanged throughout the ovarian cycle, however elevated in 11-day full-grown immature ovarian follicle and after hCG-induction. Interestingly, CREB3 expression was induced three folds on the day of spawning as well as during hCG-induced oocyte maturation. Based on the synergistic expression pattern, CREB1 is likely to control oocyte growth, whereas CREB 2 and 3 contribute to oocyte maturation in tilapia and the latter seems to be critical. In catfish, a single form of CREB showed a maximum expression during spawning phase and hCG-induced maturation both in vivo and in vitro augmented CREB expression. These results suggest that spatial and temporal expression of CREBs seems to be important for final oocyte maturation and may also regulate oocyte growth in fish. PMID:26700177

  13. Rit-mediated Stress Resistance Involves a p38-Mitogen- and Stress-activated Protein Kinase 1 (MSK1)-dependent cAMP Response Element-binding Protein (CREB) Activation Cascade*

    PubMed Central

    Shi, Geng-Xian; Cai, Weikang; Andres, Douglas A.

    2012-01-01

    The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and BclXL proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression. PMID:23038261

  14. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  15. Heat shock and deciliation induce phosphorylation of histone H1 in T. pyriformis.

    PubMed

    Glover, C V; Vavra, K J; Guttman, S D; Gorovsky, M A

    1981-01-01

    Both heat shock and decilliation of Tetrahymena pyriformis lead to an increase in the level of histone H1 phosphorylation. After heat shock, starved or growing cells reach the same maximum level of H1 phosphorylation, although the increase is more easily detected in starved cells because of their relatively low initial level of phosphorylation. In starved cells, stress-induced phosphorylation is rapid, involves a large percentage of the H1, occurs at multiple sites on the H1 molecule and is inhibited by cycloheximide. Stress-induced phosphorylation of H1 in Tetrahymena thus has many properties in common with cell-cycle-dependent H1 phosphorylation although it is not coupled to the cell cycle.

  16. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    PubMed Central

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells. PMID:26666690

  17. EGCG ameliorates high-fat- and high-fructose-induced cognitive defects by regulating the IRS/AKT and ERK/CREB/BDNF.

    PubMed

    Mi, Yashi; Qi, Guoyuan; Fan, Rong; Qiao, Qinglian; Sun, Yali; Gao, Yuqi; Liu, Xuebo

    2017-07-24

    Obesity, which is caused by an energy imbalance between calorie intake and consumption, has become a major international health burden. Obesity increases the risk of insulin resistance and age-related cognitive decline, accompanied by peripheral inflammation. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, possesses antioxidant, anti-inflammatory, and cardioprotective activities; however, few reports have focused on its potential effect on cognitive disorders. In this study, our goal was to investigate the protective effects of EGCG treatment on insulin resistance and memory impairment induced by a high-fat and high-fructose diet (HFFD). We randomly assigned 3-mo-old C57BL/6J mice to 3 groups with different diets: control group, HFFD group, and HFFD plus EGCG group. Memory loss was assessed by using the Morris water maze test, during which EGCG was observed to prevent HFFD-elicited memory impairment and neuronal loss. Consistent with these results, EGCG attenuated HFFD-induced neuronal damage. Of note, EGCG significantly ameliorated insulin resistance and cognitive disorder by up-regulating the insulin receptor substrate-1 (IRS-1)/AKT and ERK/cAMP response element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathways. Long-term HFFD-triggered neuroinflammation was restored by EGCG supplementation by inhibiting the MAPK and NF-κB pathways, as well as the expression of inflammatory mediators, such as TNF-α. EGCG also reversed high glucose and glucosamine-induced insulin resistance in SH-SY5Y neuronal cells by improving the oxidized cellular status and mitochondrial function. To our knowledge, this study is the first to provide compelling evidence that the nutritional compound EGCG has the potential to ameliorate HFFD-triggered learning and memory loss.-Mi, Y., Qi, G., Fan, R., Qiao, Q., Sun, Y., Gao, Y., Liu, X. EGCG ameliorates high-fat- and high-fructose-induced cognitive defects by regulating the IRS/AKT and

  18. Histone Acetyltransferase p300/CREB-binding Protein-associated Factor (PCAF) Is Required for All-trans-retinoic Acid-induced Granulocytic Differentiation in Leukemia Cells.

    PubMed

    Sunami, Yoshitaka; Araki, Marito; Kan, Shin; Ito, Akihiro; Hironaka, Yumi; Imai, Misa; Morishita, Soji; Ohsaka, Akimichi; Komatsu, Norio

    2017-02-17

    Differentiation therapy with all-trans-retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL); however, the molecular mechanism by which ATRA induces granulocytic differentiation remains unclear. We previously reported that the inhibition of the NAD-dependent histone deacetylase (HDAC) SIRT2 induces granulocytic differentiation in leukemia cells, suggesting the involvement of protein acetylation in ATRA-induced leukemia cell differentiation. Herein, we show that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase (HAT), is a prerequisite for ATRA-induced granulocytic differentiation in leukemia cells. We found that PCAF expression was markedly increased in leukemia cell lines (NB4 and HL-60) and primary APL cells during ATRA-induced granulocytic differentiation. Consistent with these results, the expression of PCAF was markedly up-regulated in the bone marrow cells of APL patients who received ATRA-containing chemotherapy. The knockdown of PCAF inhibited ATRA-induced granulocytic differentiation in leukemia cell lines and primary APL cells. Conversely, the overexpression of PCAF induced the expression of the granulocytic differentiation marker CD11b at the mRNA level. Acetylome analysis identified the acetylated proteins after ATRA treatment, and we found that histone H3, a known PCAF acetylation substrate, was preferentially acetylated by the ATRA treatment. Furthermore, we have demonstrated that PCAF is required for the acetylation of histone H3 on the promoter of ATRA target genes, such as CCL2 and FGR, and for the expression of these genes in ATRA-treated leukemia cells. These results strongly support our hypothesis that PCAF is induced and activated by ATRA, and the subsequent acetylation of PCAF substrates promotes granulocytic differentiation in leukemia cells. Targeting PCAF and its downstream acetylation targets could serve as a novel therapeutic strategy to overcome all subtypes of AML.

  19. The neuroprotective effects of α-iso-cubebene on dopaminergic cell death: involvement of CREB/Nrf2 signaling.

    PubMed

    Park, Sun Young; Son, Beung Gu; Park, Young Hoon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

    2014-09-01

    As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.

  20. Protein kinase C phosphorylates the cAMP response element binding protein in the hypothalamic paraventricular nucleus during morphine withdrawal

    PubMed Central

    Martín, F; Mora, L; Laorden, ML; Milanés, MV

    2011-01-01

    BACKGROUND AND PURPOSE Exposure to drugs of abuse or stress results in adaptation in the brain involving changes in gene expression and transcription factors. Morphine withdrawal modulates gene expression through various second-messenger signal transduction systems. Here, we investigated changes in activation of the transcription factor, cAMP-response element binding protein (CREB), in the hypothalamic paraventricular nucleus (PVN) and the kinases that may mediate the morphine withdrawal-triggered activation of CREB and the response of the hypothalamic-pituitary-adrenocortical (HPA) axis after naloxone-induced morphine withdrawal. EXPERIMENTAL APPROACH The effects of morphine dependence and withdrawal, phosphorylated CREB (pCREB), corticotrophin-releasing factor (CRF) expression in the PVN and HPA axis activity were measured using immunoblotting, immunohistochemistry and radioimmunoassay in controls and in morphine-dependent rats, withdrawn with naloxone and pretreated with vehicle, calphostin C, chelerythrine (inhibitors of protein kinase C (PKC) or SL-327 [inhibitor of extracellular signal regulated kinase (ERK) kinase]. In addition, changes in PKCα and PKCγ immunoreactivity were measured after 60 min of withdrawal. KEY RESULTS In morphine-withdrawn rats, pCREB immunoreactivity was increased within CRF immunoreactive neurons in the PVN and plasma corticosterone levels were raised. SL-327, at doses that reduced the augmented pERK levels in the PVN, did not attenuate the rise in pCREB immunoreactivity or plasma corticosterone secretion. In contrast, PKC inhibition reduced the withdrawal-triggered rise in pCREB, pERK1/2 and corticosterone secretion. CONCLUSIONS AND IMPLICATIONS PKC mediated, in part, both CREB activation and the HPA response to morphine withdrawal. The ERK kinase/ERK pathway might not be necessary for either activation of CREB or HPA axis hyperactivity. PMID:21615389

  1. Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1.

    PubMed Central

    Sanguinetti, Amy R; Cao, Haiming; Corley Mastick, Cynthia

    2003-01-01

    Caveolin-1 is phosphorylated on Tyr(14) in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF-/-). Using cell lines derived from single-kinase-knockout mice (Src-/-, Yes-/- and Fyn-/-), we show that expression of Fyn, but not Src or Yes, is required for stress-induced caveolin phosphorylation. Heterologous expression of Fyn in the SYF-/- and Fyn-/- cells was sufficient to reconstitute stress-induced caveolin phosphorylation, and overexpression of Fyn in wild-type cells induced hyperphosphorylation of caveolin. Fyn was autophosphorylated following oxidative stress, verifying activation of this kinase. Interestingly, there was a concomitant increase in the phosphorylation of Fyn on its Csk (C-terminal Src kinase) site, indicating feedback inhibition. Csk binds to phosphocaveolin [Cao, Courchesne and Mastick (2002) J. Biol. Chem. 277, 8771-8774] and should phosphorylate any co-localized Src-family kinases. Oxidative-stress-induced phosphorylation of caveolin-1 also requires expression of Abl [Sanguinetti and Mastick (2003) Cell Signal. 15, 289-298]. Using inhibitors and cells derived from knockout mice, we verified a requirement for both Abl and Fyn in stress-induced caveolin phosphorylation in a single cell type. Our data suggest a novel mechanism for attenuation of Src-kinase activity by Abl: stable tyrosine phosphorylation of a scaffolding protein, caveolin, and recruitment of Csk. Paxillin, a substrate of both Abl and Src, organizes a similar regulatory complex. PMID:12921535

  2. CREB influences timing and entrainment of the SCN circadian clock.

    PubMed

    Lee, Boyoung; Li, Aiqing; Hansen, Katelin F; Cao, Ruifeng; Yoon, Jae Hwa; Obrietan, Karl

    2010-12-01

    The transcriptional feedback circuit, which is at the core of the suprachiasmatic nucleus (SCN) circadian (i.e., 24 h) clock, is tightly coupled to both external entrainment cues, such as light, as well as rhythmic cues that arise on a system-wide level within the SCN. One potential signaling pathway by which these cues are conveyed to the molecular clock is the CREB/CRE transcriptional cascade. In this study, we employed a tetracycline-inducible CREB repressor mouse strain, in which approximately 60% of the SCN neurons express the transgene, to test CREB functionality in the clock and its effects on overt rhythmicity. We show that attenuated CREB signaling in the SCN led to a significant reduction in light-evoked clock entrainment. An examination of circadian timing revealed that CREB repressor mice exhibited normal free-running rhythms in the absence of external lighting cues. However, under conditions of constant light, which typically leads to a lengthening of the circadian period, CREB repressor mice exhibited a dramatic arrhythmic phenotype, which could be reversed with doxycycline. At a cellular level, the repression of CREB led to a significant reduction in both the expression of the circadian clock proteins PERIOD1 and PERIOD2 and the clock output hormones AVP and VIP. Together, these data support the idea that the CRE transcriptional pathway orchestrates transcriptional events that are essential for both the maintenance of SCN timing and light entrainment of the circadian clock.

  3. Mechanisms Underlying the Antidepressant Response of Acupuncture via PKA/CREB Signaling Pathway

    PubMed Central

    Jiang, Huili; Zhang, Xuhui; Wang, Yu; Zhang, Huimin; Li, Jing; Yang, Xinjing; Zhao, Bingcong; Zhang, Chuntao; Yu, Miao; Xu, Mingmin; Yu, Qiuyun; Liang, Xingchen; Li, Xiang; Shi, Peng

    2017-01-01

    Protein kinase A (PKA)/cAMP response element-binding (CREB) protein signaling pathway, contributing to impaired neurogenesis parallel to depressive-like behaviors, has been identified as the crucial factor involved in the antidepressant response of acupuncture. However, the molecular mechanisms associated with antidepressant response of acupuncture, neurogenesis, and depressive-like behaviors ameliorating remain unexplored. The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor H89 in in vivo experiments. Our results indicated that the expression of hippocampal PKA-α and p-CREB was significantly downregulated by chronic unpredicted mild stress (CUMS) procedures. Importantly, acupuncture reversed the downregulation of PKA-α and p-CREB. The expression of PKA-α was upregulated by fluoxetine, but not p-CREB. No significant difference was found between Acu and FLX groups on the expression of PKA-α and p-CREB. Interestingly, H89 inhibited the effects of acupuncture or fluoxetine on upregulating the expression of p-CREB, but not PKA-α. There was no significant difference in expression of CREB among the groups. Conclusively, our findings further support the hypothesis that acupuncture could ameliorate depressive-like behaviors by regulating PKA/CREB signaling pathway, which might be mainly mediated by regulating the phosphorylation level of CREB. PMID:28523193

  4. Stress-induced inhibition of translation independently of eIF2α phosphorylation.

    PubMed

    Knutsen, Jon Halvor Jonsrud; Rødland, Gro Elise; Bøe, Cathrine Arnason; Håland, Tine Weise; Sunnerhagen, Per; Grallert, Beáta; Boye, Erik

    2015-12-01

    Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution. © 2015. Published by The Company of Biologists Ltd.

  5. Stress-induced inhibition of translation independently of eIF2α phosphorylation

    PubMed Central

    Knutsen, Jon Halvor Jonsrud; Rødland, Gro Elise; Bøe, Cathrine Arnason; Håland, Tine Weise; Sunnerhagen, Per; Grallert, Beáta; Boye, Erik

    2015-01-01

    ABSTRACT Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution. PMID:26493332

  6. Myosin light chain phosphorylation is correlated with cold-induced changes in platelet shape.

    PubMed

    Kawakami, H; Higashihara, M; Ohsaka, M; Miyazaki, K; Ikebe, M; Hirano, H

    2001-12-01

    Chilling induces shape changes in platelets from disks to spheres with abundant filopodia. Such changes were time-dependent and correlated well with the phosphorylation of 20-kDa myosin light chain (LC20). Both the shape changes and the phosphorylation were reversible. After the platelets had been chilled, myosin became incorporated into the Triton X-insoluble fraction. When the chilled platelets were immunocytochemically stained, anti-myosin antibody was localized with filamentous structures inside the filopodia. These results suggest that LC20 phosphorylation and subsequent interactions with actin filaments play a crucial role in the cold-induced changes in platelet shape and in the formation of filopodia.

  7. CREB Overexpression: A Feature Associated with Negative Prognosis in Never-Smokers with NSCLC

    PubMed Central

    Seo, Hye-Sook; Liu, Diane D.; Bekele, B. Nebiyou; Kim, Mi-Kyoung; Pisters, Katherine; Lippman, Scott M.; Wistuba, Ignacio I.; Koo, Ja Seok

    2010-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. Recent advances in targeted therapies hold promise for the development of new treatments for certain subsets of cancer patients by targeting specific signaling molecule. Based on the identification of the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) as an important regulator of growth of several types of cancers and our recent findings of its importance in normal differentiation of bronchial epithelial cells, we hypothesized that CREB plays an important pathobiologic role in lung carcinogenesis. We conducted this initial study to determine whether the expression and activation status of CREB are altered in non-small cell lung cancer (NSCLC) and of any prognostic importance in NSCLC patients. We found that the expression levels of mRNA and protein of CREB and phosphorylated CREB (p-CREB) were significantly higher in most of the NSCLC cell lines and tumor specimens than in the normal human tracheobronchial epithelial (NHTBE) cells and adjacent normal lung tissue, respectively. Analysis of CREB mRNA expression and the CREB gene copy number showed that CREB overexpression occurred mainly at the transcriptional level. Immunohistochemical analysis of tissue microarray (TMA) slides containing sections of NSCLC specimens obtained from 310 patients showed that a decreased survival duration was significantly associated with overexpression of CREB or p-CREB in never-smokers but not in current or former smokers with NSCLC. These are the first reported results illustrating the potential of CREB as a molecular target for the prevention and treatment of NSCLC, especially in never-smokers. PMID:18676828

  8. Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

    2015-06-15

    In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein-nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. α-Melanocyte-stimulating hormone ameliorates ocular surface dysfunctions and lesions in a scopolamine-induced dry eye model via PKA-CREB and MEK-Erk pathways

    PubMed Central

    Ru, Yusha; Huang, Yue; Liu, Huijuan; Du, Juan; Meng, Zhu; Dou, Zexia; Liu, Xun; Wei, Rui Hua; Zhang, Yan; Zhao, Shaozhen

    2015-01-01

    Dry eye is a highly prevalent, chronic, and multifactorial disease that compromises quality of life and generates socioeconomic burdens. The pathogenic factors of dry eye disease (DED) include tear secretion abnormalities, tear film instability, and ocular surface inflammation. An effective intervention targeting the pathogenic factors is needed to control this disease. Here we applied α-Melanocyte-stimulating hormone (α-MSH) twice a day to the ocular surface of a scopolamine-induced dry eye rat model. The results showed that α-MSH at different doses ameliorated tear secretion, tear film stability, and corneal integrity, and corrected overexpression of proinflammatory factors, TNF-α, IL-1β, and IFN-γ, in ocular surface of the dry eye rats. Moreover, α-MSH, at 10−4 μg/μl, maintained corneal morphology, inhibited apoptosis, and restored the number and size of conjunctival goblet cells in the dry eye rats. Mechanistically, α-MSH activated both PKA-CREB and MEK-Erk pathways in the dry eye corneas and conjunctivas; pharmacological blockade of either pathway abolished α-MSH’s protective effects, suggesting that both pathways are necessary for α-MSH’s protection under dry eye condition. The peliotropic protective functions and explicit signaling mechanism of α-MSH warrant translation of the α-MSH-containing eye drop into a novel and effective intervention to DED. PMID:26685899

  10. Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB

    PubMed Central

    Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

    2015-01-01

    In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein–nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci. PMID:25662274

  11. α-Melanocyte-stimulating hormone ameliorates ocular surface dysfunctions and lesions in a scopolamine-induced dry eye model via PKA-CREB and MEK-Erk pathways.

    PubMed

    Ru, Yusha; Huang, Yue; Liu, Huijuan; Du, Juan; Meng, Zhu; Dou, Zexia; Liu, Xun; Wei, Rui Hua; Zhang, Yan; Zhao, Shaozhen

    2015-12-21

    Dry eye is a highly prevalent, chronic, and multifactorial disease that compromises quality of life and generates socioeconomic burdens. The pathogenic factors of dry eye disease (DED) include tear secretion abnormalities, tear film instability, and ocular surface inflammation. An effective intervention targeting the pathogenic factors is needed to control this disease. Here we applied α-Melanocyte-stimulating hormone (α-MSH) twice a day to the ocular surface of a scopolamine-induced dry eye rat model. The results showed that α-MSH at different doses ameliorated tear secretion, tear film stability, and corneal integrity, and corrected overexpression of proinflammatory factors, TNF-α, IL-1β, and IFN-γ, in ocular surface of the dry eye rats. Moreover, α-MSH, at 10(-4) μg/μl, maintained corneal morphology, inhibited apoptosis, and restored the number and size of conjunctival goblet cells in the dry eye rats. Mechanistically, α-MSH activated both PKA-CREB and MEK-Erk pathways in the dry eye corneas and conjunctivas; pharmacological blockade of either pathway abolished α-MSH's protective effects, suggesting that both pathways are necessary for α-MSH's protection under dry eye condition. The peliotropic protective functions and explicit signaling mechanism of α-MSH warrant translation of the α-MSH-containing eye drop into a novel and effective intervention to DED.

  12. Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation.

    PubMed

    Röhrig, Horst; Schmidt, Jürgen; Colby, Thomas; Bräutigam, Anne; Hufnagel, Peter; Bartels, Dorothea

    2006-08-01

    Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.

  13. PAK-dependent STAT5 serine phosphorylation is required for BCR-ABL-induced leukemogenesis

    PubMed Central

    Berger, A; Hoelbl-Kovacic, A; Bourgeais, J; Hoefling, L; Warsch, W; Grundschober, E; Uras, I Z; Menzl, I; Putz, E M; Hoermann, G; Schuster, C; Fajmann, S; Leitner, E; Kubicek, S; Moriggl, R; Gouilleux, F; Sexl, V

    2014-01-01

    The transcription factor STAT5 (signal transducer and activator of transcription 5) is frequently activated in hematological malignancies and represents an essential signaling node downstream of the BCR-ABL oncogene. STAT5 can be phosphorylated at three positions, on a tyrosine and on the two serines S725 and S779. We have investigated the importance of STAT5 serine phosphorylation for BCR-ABL-induced leukemogenesis. In cultured bone marrow cells, expression of a STAT5 mutant lacking the S725 and S779 phosphorylation sites (STAT5SASA) prohibits transformation and induces apoptosis. Accordingly, STAT5SASA BCR-ABL+ cells display a strongly reduced leukemic potential in vivo, predominantly caused by loss of S779 phosphorylation that prevents the nuclear translocation of STAT5. Three distinct lines of evidence indicate that S779 is phosphorylated by group I p21-activated kinase (PAK). We show further that PAK-dependent serine phosphorylation of STAT5 is unaffected by BCR-ABL tyrosine kinase inhibitor treatment. Interfering with STAT5 phosphorylation could thus be a novel therapeutic approach to target BCR-ABL-induced malignancies. PMID:24263804

  14. FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5.

    PubMed

    Li, Weiquan; Aurandt, Jennifer; Jürgensen, Claudia; Jürgense, Claudia; Rao, Yi; Guan, Kun-Liang

    2006-01-01

    During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.

  15. Metformin inhibits StAR expression in human endometriotic stromal cells via AMPK-mediated disruption of CREB-CRTC2 complex formation.

    PubMed

    Xu, Jia-Ning; Zeng, Cheng; Zhou, Yan; Peng, Chao; Zhou, Ying-Fang; Xue, Qing

    2014-08-01

    Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production. The aim of this study was to investigate the molecular and cellular mechanism by which metformin regulates StAR expression in human endometriotic stromal cells (ESCs). ESCs derived from ovarian endometriomas were cultured with metformin and prostaglandin E2 (PGE2). StAR mRNA was measured by quantitative PCR; pregnenolone, progesterone, and estrogen production were measured by ELISA kits; steroidogenic acute regulatory protein (StAR), AMP-activated protein kinase, cAMP response element binding protein (CREB), and CREB-regulated transcription coactivator 2 (CRTC2) protein expression were measured by Western blot assay; and CRTC2 translocation and its association with CREB were assessed by coimmunoprecipitation assay and CRTC2-CREB complex binding by a chromatin immunoprecipitation assay. 1) StAR mRNA levels in ESCs are 264 times higher than those in endometrial cells. 2) Metformin downregulates the StAR mRNA expression (maximum 31.7%) stimulated by PGE2 (2.4-fold) in ESCs. 3) PGE2 induces CRTC2 translocation and enhances its association with CREB to form a transcription complex that binds to the StAR promoter region. 4) Metformin prevents the nuclear translocation of CRTC2 by increasing AMP-activated protein kinase phosphorylation. This inhibits transcription of StAR by disrupting formation of the CREB-CRTC2 complex, involved in activation of the StAR promoter cAMP response element. We have demonstrated a detailed mechanistic analysis of StAR expression regulated by metformin in ESCs. Our data highlight a role for CRTC2 in the mechanism by which metformin inhibits StAR expression.

  16. Acetylcholine-induced phosphorylation in isolated outer hair cells.

    PubMed

    Szõnyi, M; Csermely, P; Sziklai, I

    1999-03-01

    Two groups of isolated, surviving outer hair cells (OHCs) of guinea pig cochleas (n = 20, for each group) were treated with 10 microM acetylcholine or acetylcholine plus strichnine (an alpha9 nAChR antagonist), respectively, under short-term tissue culture conditions. The protein content of the cell homogenates was separated by SDS-polyacrylamide gel electrophoresis, Western blotted and labelled with an antibody against phosphoserine residues. Signals were detected using the ECL system. Acetylcholine challenge of the OHCs resulted in a difference in the pattern of phosphorylated proteins from those of strichnine pretreated cells. A 220 kDa and a 120 kDa protein expressed a more intense phosphorylated state in the ACh group compared with the ACh plus strichnine group. The 220 kDa phosphoprotein is in the range of the cytoskeletal protein beta-fodrin, whereas the 120 kDa fraction is similar to alpha-fodrin or an ankyrin isoform. Phosphorylation of proteins due to activation of the AChR by agonist can play a role in the signalling mechanism between receptor activation and increase in the electromotile capability of isolated OHCs.

  17. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.

    PubMed

    Liu, Siqi; Cai, Xin; Wu, Jiaxi; Cong, Qian; Chen, Xiang; Li, Tuo; Du, Fenghe; Ren, Junyao; Wu, You-Tong; Grishin, Nick V; Chen, Zhijian J

    2015-03-13

    During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway. Copyright © 2015, American Association for the Advancement of Science.

  18. Γ-Ionizing radiation-induced activation of the EGFR-p38/ERK-STAT3/CREB-1-EMT pathway promotes the migration/invasion of non-small cell lung cancer cells and is inhibited by podophyllotoxin acetate.

    PubMed

    Cho, Jeong Hyun; Hong, Wan Gi; Jung, Yu-Jin; Lee, Jaeseok; Lee, Eunah; Hwang, Sang-Gu; Um, Hong-Duck; Park, Jong Kuk

    2016-06-01

    Here, we report a new intracellular signaling pathway involved in γ-ionizing radiation (IR)-induced migration/invasion and show that podophyllotoxin acetate (PA) inhibits the IR-induced invasion and migration of A549 cells (a non-small cell lung cancer (NSCLC) cell line). Our results revealed that IR increased the invasion/migration of A549 cells, and this effect was decreased by 10 nM PA treatment. PA also inhibited the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). The IR-induced increases in invasion/migration were associated with the activation of EGFR-AKT, and PA inhibited this effect. P38 and p44/42 ERK were also involved in IR-induced invasion/migration, and combined treatments with PA plus inhibitors of each MAPK synergistically blocked this invasion/migration. In terms of transcription factors (TFs), IR-induced increases in cyclic AMP response element-binding protein-1 (CREB-1) and signal transducer and activator of transcription 3 (STAT3) increased invasion/migration and EMT. PA also inhibited these transcription factors and then blocked IR-induced invasion/migration. Collectively, these results indicate that IR induces cancer cell invasion/migration by activating the EGFR-p38/ERK-CREB-1/STAT3-EMT pathway and that PA blocks this pathway to inhibit IR-induced invasion/migration.

  19. Neuroprotective effect of arctigenin via upregulation of P-CREB in mouse primary neurons and human SH-SY5Y neuroblastoma cells.

    PubMed

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-09-10

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

  20. Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-01-01

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB. PMID:24025424

  1. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    PubMed

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

  2. Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation

    PubMed Central

    2011-01-01

    Introduction Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation. Methods IR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated. Results The average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked

  3. Dexmedetomidine-Induced Contraction Involves CPI-17 Phosphorylation in Isolated Rat Aortas

    PubMed Central

    Ok, Seong-Ho; Kwon, Seong-Chun; Baik, Jiseok; Hong, Jeong-Min; Oh, Jiah; Han, Jeong Yeol; Sohn, Ju-Tae

    2016-01-01

    Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca2+]i) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca2+]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC. PMID:27706026

  4. Dexmedetomidine-Induced Contraction Involves CPI-17 Phosphorylation in Isolated Rat Aortas.

    PubMed

    Ok, Seong-Ho; Kwon, Seong-Chun; Baik, Jiseok; Hong, Jeong-Min; Oh, Jiah; Han, Jeong Yeol; Sohn, Ju-Tae

    2016-09-30

    Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca(2+)]i) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca(2+)]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC.

  5. Acid activation of Trpv1 leads to an up-regulation of calcitonin gene-related peptide expression in dorsal root ganglion neurons via the CaMK-CREB cascade: a potential mechanism of inflammatory pain.

    PubMed

    Nakanishi, Masako; Hata, Kenji; Nagayama, Tomotaka; Sakurai, Teruhisa; Nishisho, Toshihiko; Wakabayashi, Hiroki; Hiraga, Toru; Ebisu, Shigeyuki; Yoneda, Toshiyuki

    2010-08-01

    Increased production of calcitonin gene-related peptide (CGRP) in sensory neurons is implicated in inflammatory pain. The inflammatory site is acidic due to proton release from infiltrating inflammatory cells. Acid activation of peripheral nociceptors relays pain signals to the CNS. Here, we examined whether acid activated the transient receptor potential vanilloid subtype 1 (Trpv1), a widely recognized acid-sensing nociceptor and subsequently increased CGRP expression. Chemically induced inflammation was associated with thermal hyperalgesia and increased CGRP expression in dorsal root ganglion (DRG) in rats. In organ cultures of DRG, acid (pH 5.5) elevated CGRP expression and the selective Trpv1 antagonist 5'-Iodoresiniferatoxin decreased it. Trpv1-deficient DRG showed reduced CGRP increase by acid. Of note, many of CGRP/Trpv1-positive DRG neurons exhibited the phosphorylation of cAMP response element-binding protein (CREB), a nociceptive transcription factor. Knockdown of CREB by small interfering RNA or a dominant-negative form of CREB diminished acid-elevated CGRP expression. Acid elevated the transcriptional activity of CREB, which in turn stimulated CGRP gene promoter activity. These effects were inhibited by a Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitor KN-93. In conclusion, our results suggest that inflammatory acidic environments activate Trpv1, leading to an up-regulation of CGRP expression via CaMK-CREB cascade, a series of events that may be associated with inflammatory pain.

  6. Resveratrol ameliorates spatial learning memory impairment induced by Aβ1-42 in rats.

    PubMed

    Wang, Rui; Zhang, Yu; Li, Jianguo; Zhang, Ce

    2017-03-06

    β-amyloid (Aβ) deposition is considered partially responsible for cognitive dysfunction in Alzheimer's disease (AD). Recently, resveratrol has been reported to play a potential role as a neuroprotective biofactor by modulating Aβ pathomechanisms, including through anti-neuronal apoptotic, anti-oxidative stress, and anti-neuroinflammatory effects. In addition, SIRT1 has been demonstrated to modulate learning and memory function by regulating the expression of cAMP response binding protein (CREB), which involves in modulating the expression of SIRT1. However, whether resveratrol can alleviate Aβ-induced cognitive dysfunction, whether SIRT1 expression and CREB phosphorylation in the hippocampus are affected by Aβ, and whether resveratrol influences these effects remain unknown. In the present study, we used a hippocampal injection model in rats to investigate the effects of resveratrol on Aβ1-42-induced impairment of spatial learning, memory and synaptic plasticity as well as on alterations of SIRT1 expression and CREB phosphorylation. We found that resveratrol significantly reversed the water maze behavioral impairment and the attenuation of long-term potentiation (LTP) in area CA1 that were induced by hippocampal injection of Aβ1-42. Interestingly, resveratrol also prevented the Aβ1-42-induced reductions in SIRT1 expression and CREB phosphorylation in rat hippocampus. In conclusion, in rats, resveratrol protects neurons against Aβ1-42-induced disruption of spatial learning, memory and hippocampal LTP. The mechanisms underlying the neuroprotective effects may involve rescue of SIRT1 expression and CREB phosphorylation.

  7. Aluminum induces neurofilament aggregation by stabilizing cross-bridging of phosphorylated c-terminal sidearms.

    PubMed

    Kushkuley, Jacob; Metkar, Shailesh; Chan, Walter K-H; Lee, Sangmook; Shea, Thomas B

    2010-03-31

    Exposure to neurotoxin aluminum neurotoxicity is accompanied by the perikaryal accumulation of tangles of phosphorylated neurofilaments (NFs). We examined their formation and reversibility under cell-free conditions. AlCl3 induced dose-dependent formation of NF aggregates, ultimately incorporating 100% of detectable NFs. The same concentration of CaCl2 induced approximately 25% of NFs to form longitudinal dimers and did not induce aggregation. AlCl3 induced similar percentages of aggregates in the presence or absence of CaCl2, and CaCl2 could not reduce pre-formed aggregates. CaCl(2)-induced dimers and AlCl(3)-induced aggregates were prevented by prior NF dephosphorylation. While CaCl(2)-induced dimers were dissociated by phosphatase treatment, AlCl(3)-induced aggregates were only reduced by approximately 50%, suggesting that aggregates may sequester phosphorylation sites. Since phosphatases regulate NF phosphorylation within perikarya, inhibition of NF dephosphorylation by aluminum would promote perikaryal NF phosphorylation and foster precocious phospho-dependent NF-NF associations. These findings are consistent with the notion that prolonged interactions induced among phospho-NFs by the trivalent aluminum impairs axonal transport and promotes perikaryal aggregation. Copyright 2010. Published by Elsevier B.V.

  8. Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified

    PubMed Central

    Hart, Katherine; Kothari, Anisha; Mackintosh, Samuel G.; Kovak, Matthew R.; Chambers, Timothy C.

    2016-01-01

    Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model. PMID:27738316

  9. Uncoupling Stress-Inducible Phosphorylation of Heat Shock Factor 1 from Its Activation

    PubMed Central

    Budzyński, Marek A.; Puustinen, Mikael C.; Joutsen, Jenny

    2015-01-01

    In mammals the stress-inducible expression of genes encoding heat shock proteins is under the control of the heat shock transcription factor 1 (HSF1). Activation of HSF1 is a multistep process, involving trimerization, acquisition of DNA-binding and transcriptional activities, which coincide with several posttranslational modifications. Stress-inducible phosphorylation of HSF1, or hyperphosphorylation, which occurs mainly within the regulatory domain (RD), has been proposed as a requirement for HSF-driven transcription and is widely used for assessing HSF1 activation. Nonetheless, the contribution of hyperphosphorylation to the activity of HSF1 remains unknown. In this study, we generated a phosphorylation-deficient HSF1 mutant (HSF1Δ∼PRD), where the 15 known phosphorylation sites within the RD were disrupted. Our results show that the phosphorylation status of the RD does not affect the subcellular localization and DNA-binding activity of HSF1. Surprisingly, under stress conditions, HSF1Δ∼PRD is a potent transactivator of both endogenous targets and a reporter gene, and HSF1Δ∼PRD has a reduced activation threshold. Our results provide the first direct evidence for uncoupling stress-inducible phosphorylation of HSF1 from its activation, and we propose that the phosphorylation signature alone is not an appropriate marker for HSF1 activity. PMID:25963659

  10. The Myb-p300-CREB axis modulates intestine homeostasis, radiosensitivity and tumorigenesis

    PubMed Central

    Sampurno, S; Bijenhof, A; Cheasley, D; Xu, H; Robine, S; Hilton, D; Alexander, W S; Pereira, L; Mantamadiotis, T; Malaterre, J; Ramsay, R G

    2013-01-01

    The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300–Myb–CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI

  11. Downregulation of CREB expression in Alzheimer's brain and in Aβ-treated rat hippocampal neurons

    PubMed Central

    2011-01-01

    Background Oxidative stress plays an important role in neuronal dysfunction and neuron loss in Alzheimer's brain. Previous studies have reported downregulation of CREB-mediated transcription by oxidative stress and Aβ. The promoter for CREB itself contains cyclic AMP response elements. Therefore, we examined the expression of CREB in the hippocampal neurons of Tg2576 mice, AD post-mortem brain and in cultured rat hippocampal neurons exposed to Aβ aggregates. Results Laser Capture Microdissection of hippocampal neurons from Tg2576 mouse brain revealed decreases in the mRNA levels of CREB and its target, BDNF. Immunohistochemical analysis of Tg2576 mouse brain showed decreases in CREB levels in hippocampus and cortex. Markers of oxidative stress were detected in transgenic mouse brain and decreased CREB staining was observed in regions showing abundance of astrocytes. There was also an inverse correlation between SDS-extracted Aβ and CREB protein levels in Alzheimer's post-mortem hippocampal samples. The levels of CREB-regulated BDNF and BIRC3, a caspase inhibitor, decreased and the active cleaved form of caspase-9, a marker for the intrinsic pathway of apoptosis, was elevated in these samples. Exposure of rat primary hippocampal neurons to Aβ fibrils decreased CREB promoter activity. Decrease in CREB mRNA levels in Aβ-treated neurons was reversed by the antioxidant, N-acetyl cysteine. Overexpression of CREB by adenoviral transduction led to significant protection against Aβ-induced neuronal apoptosis. Conclusions Our findings suggest that chronic downregulation of CREB-mediated transcription results in decrease of CREB content in the hippocampal neurons of AD brain which may contribute to exacerbation of disease progression. PMID:21854604

  12. Activation of cAMP-response-element-binding protein (CREB) after focal cerebral ischemia stimulates neurogenesis in the adult dentate gyrus

    PubMed Central

    Zhu, Dong Ya; Lau, Lorraine; Liu, Shu Hong; Wei, Jian She; Lu, You Ming

    2004-01-01

    New neurons are generated in adult mammalians and may contribute to repairing the brain after injury. Here, we show that the number of new neurons in the dentate gyrus of adult rats increased in cerebral ischemic stroke and correlated with activation of the cAMP-response-element-binding protein (CREB). Inhibition of endogenous CREB by expression of a dominant-negative mutant of CREB (CREB-S133A or CREB-R287L) blocked ischemia-induced neurogenesis in the dentate gyrus of adult rats, whereas expression of constitutively active CREB, VP16-CREB, increased the number of new neurons. Thus, our findings provide roles and regulatory mechanisms for CREB in adult neurogenesis and possibly suggest a practical strategy for replacing dead neurons in brain injury. PMID:15197280

  13. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

    PubMed Central

    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  14. Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells.

    PubMed

    Choi, Yoon Jung; Kim, So Young; Oh, Jung Min; Juhnn, Yong Sung

    2009-08-31

    Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.

  15. Phosphorylated tau potentiates Aβ-induced mitochondrial damage in mature neurons.

    PubMed

    Quintanilla, Rodrigo A; von Bernhardi, Rommy; Godoy, Juan A; Inestrosa, Nibaldo C; Johnson, Gail V W

    2014-11-01

    Tau phosphorylated at the PHF-1 epitope (S396/S404) is likely involved in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which tau phosphorylated at these sites negatively impacts neuronal functions are still under scrutiny. Previously, we showed that expression of tau truncated at D421 enhances mitochondrial dysfunction induced by Aβ in cortical neurons. To extend these findings, we expressed tau pseudo-phosphorylated at S396/404 (T42EC) in mature and young cortical neurons and evaluated different aspects of mitochondrial function in response to Aβ. Expression of T42EC did not induce significant changes in mitochondrial morphology, mitochondrial length, or mitochondrial transport, compared to GFP and full-length tau. However, T42EC expression enhanced Aβ-induced mitochondrial membrane potential loss and increased superoxide levels compared to what was observed in mature neurons expressing full-length tau. The same effect was observed in mature neurons that expressed both pseudo-phosphorylated and truncated tau when they were treated with Aβ. Interestingly, the mitochondrial failure induced by Aβ in mature neurons that expressed T42EC, was not observed in young neurons expressing T42EC. These novel findings suggest that phosphorylated tau (PHF-1 epitope) enhances Aβ-induced mitochondrial injury, which contributes to neuronal dysfunction and to the pathogenesis of AD. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Rapamycin induces Bad phosphorylation in association with its resistance to human lung cancer cells.

    PubMed

    Liu, Yan; Sun, Shi-Yong; Owonikoko, Taofeek K; Sica, Gabriel L; Curran, Walter J; Khuri, Fadlo R; Deng, Xingming

    2012-01-01

    Inhibition of mTOR signaling by rapamycin has been shown to activate extracellular signal-regulated kinase 1 or 2 (ERK1/2) and Akt in various types of cancer cells, which contributes to rapamycin resistance. However, the downstream effect of rapamycin-activated ERKs and Akt on survival or death substrate(s) remains unclear. We discovered that treatment of human lung cancer cells with rapamycin results in enhanced phosphorylation of Bad at serine (S) 112 and S136 but not S155 in association with activation of ERK1/2 and Akt. A higher level of Bad phosphorylation was observed in rapamycin-resistant cells compared with parental rapamycin-sensitive cells. Thus, Bad phosphorylation may contribute to rapamycin resistance. Mechanistically, rapamycin promotes Bad accumulation in the cytosol, enhances Bad/14-3-3 interaction, and reduces Bad/Bcl-XL binding. Rapamycin-induced Bad phosphorylation promotes its ubiquitination and degradation, with a significant reduction of its half-life (i.e., from 53.3-37.5 hours). Inhibition of MEK/ERK by PD98059 or depletion of Akt by RNA interference blocks rapamycin-induced Bad phosphorylation at S112 or S136, respectively. Simultaneous blockage of S112 and S136 phosphorylation of Bad by PD98059 and silencing of Akt significantly enhances rapamycin-induced growth inhibition in vitro and synergistically increases the antitumor efficacy of rapamycin in lung cancer xenografts. Intriguingly, either suppression of Bad phosphorylation at S112 and S136 sites or expression of the nonphosphorylatable Bad mutant (S112A/S136A) can reverse rapamycin resistance. These findings uncover a novel mechanism of rapamycin resistance, which may promote the development of new strategies for overcoming rapamycin resistance by manipulating Bad phosphorylation at S112 and S136 in human lung cancer.

  17. Rapamycin Induces Bad Phosphorylation in Association with Its Resistance to Human Lung Cancer Cells

    PubMed Central

    Liu, Yan; Sun, Shi-Yong; Owonikoko, Taofeek K.; Sica, Gabriel L.; Curran, Walter J.; Khuri, Fadlo R.; Deng, Xingming

    2011-01-01

    Inhibition of mTOR signaling by rapamycin has been demonstrated to activate ERK1/2 and Akt in various types of cancer cells, which contributes to rapamycin resistance. However, the downstream effect of rapamycin-activated ERKs and Akt on survival or death substrate(s) remains unclear. We discovered that treatment of human lung cancer cells with rapamycin results in enhanced phosphorylation of Bad at serine (S) 112 and S136 but not S155 in association with activation of ERK1/2 and Akt. A higher level of Bad phosphorylation was observed in rapamycin-resistant cells compared to parental rapamycin-sensitive cells. Thus, Bad phosphorylation may contribute to rapamycin resistance. Mechanistically, rapamycin promotes Bad accumulation in the cytosol, enhances Bad/14-3-3 interaction and reduces Bad/Bcl-XL binding. Rapamycin-induced Bad phosphorylation promotes its ubiquitination and degradation, with a significant reduction of its half-life (i.e. from 53.3 h to 37.5 h). Inhibition of MEK/ERK by PD98059 or depletion of Akt by RNA interference blocks rapamycin-induced Bad phosphorylation at S112 or S136, respectively. Simultaneous blockage of S112 and S136 phosphorylation of Bad by PD98059 and silencing of Akt significantly enhances rapamycin-induced growth inhibition in vitro and synergistically increases the anti-tumor efficacy of rapamycin in lung cancer xenografts. Intriguingly, either suppression of Bad phosphorylation at S112 and S136 sites or expression of the non-phosphorylatable Bad mutant (S112A/S136A) can reverse rapamycin resistance. These findings uncover a novel mechanism of rapamycin resistance, which may promote the development of new strategies for overcoming rapamycin resistance by manipulating Bad phosphorylation at S112 and S136 in human lung cancer. PMID:22057915

  18. Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry.

    PubMed

    Zolodz, Melissa D; Wood, Karl V

    2003-03-01

    Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.

  19. N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation

    PubMed Central

    Aubol, Brandon E.; Plocinik, Ryan M.; Keshwani, Malik M.; McGlone, Maria L.; Hagopian, Jonathan C.; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein. PMID:24869919

  20. Ethacrynic Acid Inhibits Sphingosylphosphorylcholine-Induced Keratin 8 Phosphorylation and Reorganization via Transglutaminase-2 Inhibition.

    PubMed

    Byun, Hyun Jung; Kang, Kyung Jin; Park, Mi Kyung; Lee, Hye Ja; Kang, June Hee; Lee, Eun Ji; Kim, You Ri; Kim, Hyun Ji; Kim, Young Woo; Jung, Kyung Chae; Kim, Soo Youl; Lee, Chang Hoon

    2013-09-30

    Sphingosylphosphorylcholine (SPC) is significantly increased in the malicious ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments in PANC-1 cells. The reorganization contributes to the viscoelasticity of metastatic cancer cells resulting in increased migration. Recently, we reported that transglutaminase-2 (Tgase-2) is involved in SPC-induced K8 phosphorylation and reorganization. However, effects of Tgase-2 inhibitors on SPC-induced K8 phosphorylation and reorganization were not clearly studied. We found that ethacrynic acid (ECA) concentration-dependently inhibited Tgase-2. Therefore, we examined the effects of ECA on SPC-induced K8 phosphorylation and reorganization. ECA concentration-dependently suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8. ECA also suppressed the SPC-induced migration and invasion. SPC induced JNK activation through Tgase-2 expression and ECA suppressed the activation and expression of JNK in PANC-1 cells. These results suggested that ECA might be useful to control Tgase-2 dependent metastasis of cancer cells such as pancreatic cancer and lung cancers.

  1. Neuroinflammation is not a Prerequisite for Diabetes-induced Tau Phosphorylation.

    PubMed

    van der Harg, Judith M; Eggels, Leslie; Ruigrok, Silvie R; Hoozemans, Jeroen J M; la Fleur, Susanne E; Scheper, Wiep

    2015-01-01

    Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD). AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM) is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS) diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD.

  2. Localized α4 Integrin Phosphorylation Directs Shear Stress-Induced Endothelial Cell Alignment

    PubMed Central

    Goldfinger, Lawrence E.; Tzima, Eleni; Stockton, Rebecca; Kiosses, William B.; Kinbara, Kayoko; Tkachenko, Eugene; Gutierrez, Edgar; Groisman, Alex; Nguyen, Phu; Chien, Shu; Ginsberg1, Mark H.

    2009-01-01

    Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to athero-protection. Here we report that localized α4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial α4 integrins became phosphorylated on Ser988. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced α4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a non-phosphorylatable α4(S988A) mutation failed to align in response to shear stress, thus establishing α4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent α4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress. PMID:18583710

  3. Leptin signaling plays a critical role in the geniposide-induced decrease of tau phosphorylation.

    PubMed

    Liu, Jianhui; Liu, Zixuan; Zhang, Yonglan; Yin, Fei

    2015-12-01

    We have previously demonstrated that geniposide attenuates the production of Aβ1-42 both in vitro and in vivo via enhancing leptin receptor signaling. But the role played by geniposide in the phosphorylation of tau and its underlying molecular mechanisms remain unclear. In this study, we investigated the effect of geniposide on the phosphorylation of tau and the role of leptin signaling in this process. Our data suggested that, accompanied by the up-regulation of leptin receptor expression, geniposide significantly decreased the phosphorylation of tau in rat primary cultured cortical neurons and in APP/PS1 transgenic mice, and this geniposide-induced decrease of tau phosphorylation could be prevented by leptin antagonist (LA). Furthermore, LA also prevented the phosphorylation of Akt at Ser-473 site and GSK-3β at Ser-9 site induced by geniposide. All these results indicate that geniposide may regulate tau phosphorylation through leptin signaling, and geniposide may be a promising therapeutic compound for the treatment of Alzheimer's disease in the future.

  4. Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.

    PubMed

    Larson, Erin B; Graham, Danielle L; Arzaga, Rose R; Buzin, Nicole; Webb, Joseph; Green, Thomas A; Bass, Caroline E; Neve, Rachael L; Terwilliger, Ernest F; Nestler, Eric J; Self, David W

    2011-11-09

    Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we used viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to postacquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Downregulating endogenous CREB in NAc shell by expressing a short hairpin RNA reduced cocaine reinforcement in similar tests, while overexpression of a dominant-negative CREB(S133A) mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes.

  5. Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

    PubMed Central

    Adderley, Shaquria P.; Joshi, Chintamani N.; Martin, Danielle N.; Tulis, David Anthony

    2012-01-01

    BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASPSer239 and VASPSer157, respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASPSer239 but inhibited phosphorylation at VASPSer157. Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASPSer239 and VASPSer157, whereas PDE5 inhibition potentiated BAY-mediated increases only at VASPSer157. In comparison, 8Br-cGMP increased phosphorylation at VASPSer239 and VASPSer157 which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASPSer239 phosphorylation and inhibition of PDE3 increased phosphorylation at VASPSer239, while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASPSer157 phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders. PMID:22347188

  6. Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 beta gene.

    PubMed Central

    Tsukada, J; Saito, K; Waterman, W R; Webb, A C; Auron, P E

    1994-01-01

    A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). Although these two enhancers appear to function cooperatively in the native sequence context, they function independently as LPS-responsive elements upon removal of an interposed silencer sequence. The new enhancer is not induced by dibutyryl cyclic AMP (dbcAMP) alone but is superinduced by costimulation with LPS-dbcAMP. This pattern of induction depends upon the nature of the sequence, a composite NF-IL6-cAMP response element (CRE) binding site. This pseudosymmetrical sequence is shown to contrast with a classical symmetric CRE which responds to dbcAMP but not LPS. DNA binding studies using in vivo nuclear extract, recombinant proteins, and specific antibodies show that LPS induces the formation of two different complexes at the enhancer: (i) an NF-IL6-CREB heterodimer and (ii) a heterodimer consisting of NF-IL6 and a non-CREB, CRE-binding protein. Cotransfection studies using NF-IL6 and CREB expression vectors show that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively, depending upon its cAMP-regulated phosphorylation state. Our data demonstrate that the newly identified enhancer is a specialized LPS-responsive sequence which can be modulated by cAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers. Images PMID:7935442

  7. Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin.

    PubMed

    Li, Fu Jun; Surolia, Ranu; Li, Huashi; Wang, Zheng; Liu, Gang; Liu, Rui-Ming; Mirov, Sergey B; Athar, Mohammad; Thannickal, Victor J; Antony, Veena B

    2017-07-01

    Exposure to cadmium (Cd) has been associated with development of chronic obstructive lung disease (COPD). The mechanisms and signaling pathways whereby Cd causes pathological peribronchiolar fibrosis, airway remodeling, and subsequent airflow obstruction remain unclear. We aimed to evaluate whether low-dose Cd exposure induces vimentin phosphorylation and Yes-associated protein 1 (YAP1) activation leading to peribronchiolar fibrosis and subsequent airway remodeling. Our data demonstrate that Cd induces myofibroblast differentiation and extracellular matrix (ECM) deposition around small (<2 mm in diameter) airways. Upon Cd exposure, α-smooth muscle actin (α-SMA) expression and the production of ECM proteins, including fibronectin and collagen-1, are markedly induced in primary human lung fibroblasts. Cd induces Smad2/3 activation and the translocation of both Smad2/3 and Yes-associated protein 1 (YAP1) into the nucleus. In parallel, Cd induces AKT and cdc2 phosphorylation and downstream vimentin phosphorylation at Ser(39) and Ser(55), respectively. AKT and cdc2 inhibitors block Cd-induced vimentin fragmentation and secretion in association with inhibition of α-SMA expression, ECM deposition, and collagen secretion. Furthermore, vimentin silencing abrogates Cd-induced α-SMA expression and decreases ECM production. Vimentin-deficient mice are protected from Cd-induced peribronchiolar fibrosis and remodeling. These findings identify two specific sites on vimentin that are phosphorylated by Cd and highlight the functional significance of vimentin phosphorylation in YAP1/Smad3 signaling that mediates Cd-induced peribronchiolar fibrosis and airway remodeling. Copyright © 2017 the American Physiological Society.

  8. Dishevelled stability is positively regulated by PKCζ-mediated phosphorylation induced by Wnt agonists.

    PubMed

    Velázquez, Dora M; Castañeda-Patlán, M Cristina; Robles-Flores, Martha

    2017-07-01

    Dishevelled (Dvl) proteins are central mediators of both canonical and non-canonical Wnt signaling. It is well known that, upon Wnt stimulation, Dvl becomes phosphorylated. However, how Wnt-induced phosphorylation of Dvl is regulated and its consequences are poorly understood. Here we found that Dvl proteins are overexpressed in colon cancer cells. In addition, we found that Wnt3a treatment rapidly induces hyperphosphorylation and stabilization of Dvl2 and Dvl3. The latter can be blocked by inhibition of Protein Kinase C (PKC)α, PKCδ, and PKCζ isoforms. We also found that Wnt3a-induced phosphorylation of Dvl3 by PKCζ is required to avoid Dvl3 degradation via proteasome. This demonstrated, to our knowledge for the first time, that hyperphosphorylation of Dvl by PKCζ results in Dvl stabilization. This is clear contrast with the consequences reported to date of CK1δ/ε-mediated Dvl phosphorylation upon Wnt treatment. Mapping the interaction domain between PKCζ and Dvl3 indicated that, although the Dvl-DIX domain is required to stabilize PKCζ-phosphorylated Dvl, it is not the region phosphorylated by this kinase. Our data show that the Dvl-DEP domain, required for specific interaction with PKCζ, is the site phosphorylated by this kinase, and also probably the Dvl-C terminus. Our findings suggest a model of positive regulation of PKCζ-mediated Dvl signaling activity, to produce a strong and sustained response to Wnt3a treatment by stabilizing Dvl protein levels. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Transcription factor CREB3L1 regulates vasopressin gene expression in the rat hypothalamus.

    PubMed

    Greenwood, Mingkwan; Bordieri, Loredana; Greenwood, Michael P; Rosso Melo, Mariana; Colombari, Debora S A; Colombari, Eduardo; Paton, Julian F R; Murphy, David

    2014-03-12

    Arginine vasopressin (AVP) is a neurohypophysial hormone regulating hydromineral homeostasis. Here we show that the mRNA encoding cAMP responsive element-binding protein-3 like-1 (CREB3L1), a transcription factor of the CREB/activating transcription factor (ATF) family, increases in expression in parallel with AVP expression in supraoptic nuclei (SONs) and paraventicular nuclei (PVNs) of dehydrated (DH) and salt-loaded (SL) rats, compared with euhydrated (EH) controls. In EH animals, CREB3L1 protein is expressed in glial cells, but only at a low level in SON and PVN neurons, whereas robust upregulation in AVP neurons accompanied DH and SL rats. Concomitantly, CREB3L1 is activated by cleavage, with the N-terminal domain translocating from the Golgi, via the cytosol, to the nucleus. We also show that CREB3L1 mRNA levels correlate with AVP transcription level in SONs and PVNs following sodium depletion, and as a consequence of diurnal rhythm in the suprachiasmatic nucleus. We tested the hypothesis that CREB3L1 activates AVP gene transcription. Both full-length and constitutively active forms of CREB3L1 (CREB3L1CA) induce the expression of rat AVP promoter-luciferase reporter constructs, whereas a dominant-negative mutant reduces expression. Rat AVP promoter deletion constructs revealed that CRE-like and G-box sequences in the region between -170 and -120 bp are important for CREB3L1 actions. Direct binding of CREB3L1 to the AVP promoter was shown by chromatin immunoprecipitation both in vitro and in the SON itself. Injection of a lentiviral vector expressing CREB3L1CA into rat SONs and PVNs resulted in increased AVP biosynthesis. We thus identify CREB3L1 as a regulator of AVP transcription in the rat hypothalamus.

  10. Transcription Factor CREB3L1 Regulates Vasopressin Gene Expression in the Rat Hypothalamus

    PubMed Central

    Greenwood, Mingkwan; Bordieri, Loredana; Greenwood, Michael P.; Rosso Melo, Mariana; Colombari, Debora S. A.; Colombari, Eduardo; Paton, Julian F. R.

    2014-01-01

    Arginine vasopressin (AVP) is a neurohypophysial hormone regulating hydromineral homeostasis. Here we show that the mRNA encoding cAMP responsive element-binding protein-3 like-1 (CREB3L1), a transcription factor of the CREB/activating transcription factor (ATF) family, increases in expression in parallel with AVP expression in supraoptic nuclei (SONs) and paraventicular nuclei (PVNs) of dehydrated (DH) and salt-loaded (SL) rats, compared with euhydrated (EH) controls. In EH animals, CREB3L1 protein is expressed in glial cells, but only at a low level in SON and PVN neurons, whereas robust upregulation in AVP neurons accompanied DH and SL rats. Concomitantly, CREB3L1 is activated by cleavage, with the N-terminal domain translocating from the Golgi, via the cytosol, to the nucleus. We also show that CREB3L1 mRNA levels correlate with AVP transcription level in SONs and PVNs following sodium depletion, and as a consequence of diurnal rhythm in the suprachiasmatic nucleus. We tested the hypothesis that CREB3L1 activates AVP gene transcription. Both full-length and constitutively active forms of CREB3L1 (CREB3L1CA) induce the expression of rat AVP promoter-luciferase reporter constructs, whereas a dominant-negative mutant reduces expression. Rat AVP promoter deletion constructs revealed that CRE-like and G-box sequences in the region between −170 and −120 bp are important for CREB3L1 actions. Direct binding of CREB3L1 to the AVP promoter was shown by chromatin immunoprecipitation both in vitro and in the SON itself. Injection of a lentiviral vector expressing CREB3L1CA into rat SONs and PVNs resulted in increased AVP biosynthesis. We thus identify CREB3L1 as a regulator of AVP transcription in the rat hypothalamus. PMID:24623760

  11. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  12. Dorsal Hippocampal CREB Is Both Necessary and Sufficient for Spatial Memory

    ERIC Educational Resources Information Center

    Sekeres, Melanie J.; Neve, Rachael L.; Frankland, Paul W.; Josselyn, Sheena A.

    2010-01-01

    Although the transcription factor CREB has been widely implicated in memory, whether it is sufficient to produce spatial memory under conditions that do not normally support memory formation in mammals is unknown. We found that locally and acutely increasing CREB levels in the dorsal hippocampus using viral vectors is sufficient to induce robust…

  13. Dorsal Hippocampal CREB Is Both Necessary and Sufficient for Spatial Memory

    ERIC Educational Resources Information Center

    Sekeres, Melanie J.; Neve, Rachael L.; Frankland, Paul W.; Josselyn, Sheena A.

    2010-01-01

    Although the transcription factor CREB has been widely implicated in memory, whether it is sufficient to produce spatial memory under conditions that do not normally support memory formation in mammals is unknown. We found that locally and acutely increasing CREB levels in the dorsal hippocampus using viral vectors is sufficient to induce robust…

  14. Cigarette smoke extracts induce overexpression of the proto-oncogenic gene interleukin-13 receptor α2 through activation of the PKA-CREB signaling pathway to trigger malignant transformation of lung vascular endothelial cells and angiogenesis.

    PubMed

    Meng, Mei; Liao, Huaidong; Zhang, Bin; Pan, Yanyan; Kong, Ying; Liu, Wenming; Yang, Ping; Huo, Zihe; Cao, Zhifei; Zhou, Quansheng

    2017-02-01

    Cigarette smoking is a major cause of lung cancer. Tumor-associated endothelial cells (TAECs) play important roles in tumor angiogenesis and metastasis. However, whether cigarette smoking can trigger genesis of lung TAECs has not been reported yet. In the current study, we used lung endothelial cell (EC) lines as a model to study the pathological effect of cigarette smoke extracts (CSEs) on human lung ECs, and found that a lower dose of 4% CSEs obviously caused abnormal morphological changes in ECs, increased the permeability of endothelial monolayer, while a higher concentration of 8% CSEs caused EC apoptosis. Strikingly, CSEs induced a 117-fold overexpression of a pro-tumorigenic interleukin-13 receptor α2 gene (IL-13Rα2, also named as CT-19) through activation of the protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling pathway. A PKA specific inhibitor H89 completely abolished CSEs-induced IL-13Rα2 overexpression. The overexpression of IL-13Rα2 in lung ECs significantly increased the tumorigenic, migratory, and angiogenic capabilities of the cells, suggesting that IL-13Rα2 promotes genesis of lung TAECs. Together, our data show that CSEs activate the PKA, CREB, and IL-13Rα2 axis in lung ECs, and IL-13Rα2 promotes the malignant transformation of lung ECs and genesis of TAECs with robust angiogenic and oncogenic capabilities. Our study provides new insight into the mechanism of CSEs-triggered lung cancer angiogenesis and tumorigenesis, suggesting that the PKA-CREB-IL-13Rα2 axis is a potential target for novel anti-lung tumor angiogenesis and anti-lung cancer drug discovery.

  15. Hepatic CREB3L3 controls whole-body energy homeostasis and improves obesity and diabetes.

    PubMed

    Nakagawa, Yoshimi; Satoh, Aoi; Yabe, Sachiko; Furusawa, Mika; Tokushige, Naoko; Tezuka, Hitomi; Mikami, Motoki; Iwata, Wakiko; Shingyouchi, Akiko; Matsuzaka, Takashi; Kiwata, Shiori; Fujimoto, Yuri; Shimizu, Hidehisa; Danno, Hirosuke; Yamamoto, Takashi; Ishii, Kiyoaki; Karasawa, Tadayoshi; Takeuchi, Yoshinori; Iwasaki, Hitoshi; Shimada, Masako; Kawakami, Yasushi; Urayama, Osamu; Sone, Hirohito; Takekoshi, Kazuhiro; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

    2014-12-01

    Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor (PPAR) α promoter in an autoloop fashion and is crucial for the ligand transactivation of PPARα by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1α. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

  16. Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

    SciTech Connect

    Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

    1989-01-15

    The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.

  17. Advanced glycation end products induce moesin phosphorylation in murine brain endothelium.

    PubMed

    Li, Qiaoqin; Liu, Hongxia; Du, Jing; Chen, Bo; Li, Qiang; Guo, Xiaohua; Huang, Xuliang; Huang, Qiaobing

    2011-02-10

    Advanced glycation end products (AGEs) have been found to play an important role in the development of diabetes, and AGE levels are correlated with the severity of diabetic complications. We have demonstrated that moesin, a protein linker between actin filaments and the plasma membrane, undergoes phosphorylation of its threonine 558 residue by AGE stimulation in human dermal microvascular endothelial cells through activation of p38 and Rho kinase (ROCK) pathways. In this study, we observed in situ whether AGEs caused phosphorylation of vascular endothelial cells in the brains of AGE-stimulated mice. The animals were injected with AGE-modified mouse serum albumin (AGE-MSA) for 7 consecutive days. Immunohistochemistry was conducted to assess the phosphorylation of moesin in brain vessels. The level of moesin protein phosphorylation was also assessed in cerebral microvessels by western blotting. The effects of p38 and ROCK activation were determined by application of a p38 inhibitor (SB203580) and a ROCK inhibitor (Y27632) at 30 min before each AGE administration. The results showed specific expression of moesin in murine brain vascular endothelial cells. AGE treatment induced a significant increase of threonine 558 phosphorylation in moesin, while inhibition of p38 and ROCK remarkably attenuated the phosphorylation of moesin. The level of moesin protein phosphorylation was also increased in cerebral microvessels, along with an increased permeability of the blood-brain barrier, while inhibition of the p38 and ROCK attenuated these responses. These results demonstrate that AGEs cause the phosphorylation of moesin in murine brain microvascular endothelial cells, with p38 and ROCK being involved in this process.

  18. PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A–SIK–CRTC3 Pathway

    PubMed Central

    MacKenzie, Kirsty F.; Clark, Kristopher; Naqvi, Shaista; McGuire, Victoria A.; Nöehren, Gesa; Kristariyanto, Yosua; van den Bosch, Mirjam; Mudaliar, Manikhandan; McCarthy, Pierre C.; Pattison, Michael J.; Pedrioli, Patrick G. A.; Barton, Geoff J.; Toth, Rachel; Prescott, Alan

    2013-01-01

    The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE2, in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A–dependent pathway. Both TLR agonists and PGE2 promote the phosphorylation of the transcription factor CREB on Ser133. However, although CREB regulates IL-10 transcription, the mutation of Ser133 to Ala in the endogenous CREB gene did not prevent the ability of PGE2 to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser343, inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE2 on IL-10 production. PMID:23241891

  19. 2-Ethoxybenzamide stimulates melanin synthesis in B16F1 melanoma cells via the CREB signaling pathway.

    PubMed

    Sato, Kazuomi; Ando, Ryosuke; Kobayashi, Honoka; Nishio, Takashi

    2016-12-01

    Non-steroidal anti-inflammatory drugs are frequently used for the treatment of inflammation, pain, and fever. In this study, we found that 2-ethoxybenzamide (ETZ) significantly enhanced melanin synthesis in B16F1 melanoma cells, and also induced melanosome formation. Therefore, we investigated the mechanism by which ETZ up-regulated melanin synthesis. Western blot analysis demonstrated that ETZ increased melanogenic protein levels, except that for TRP-2. Moreover, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR analyses showed that ETZ enhanced the mRNA levels of melanogenic genes, including microphthalmia-associated transcription factor and melanocortin 1 receptor. We also observed phosphorylation of cAMP response element-binding protein (CREB) following ETZ treatment. However, ETZ did not affect intracellular cAMP levels. ERK was also activated by ETZ treatment, and melanin content was enhanced upon treatment with the specific ERK inhibitor PD98059. Together, our results indicate that ETZ induces melanin synthesis via CREB phosphorylation.

  20. RACK1 promotes maintenance of morphine-associated memory via activation of an ERK-CREB dependent pathway in hippocampus.

    PubMed

    Liu, Litao; Zhu, Jiejun; Zhou, Liming; Wan, Lihong

    2016-02-02

    Existence of long-term drug-associated memories may be a crucial factor in drug cravings and relapse. RACK1 plays a critical role in morphine-induced reward. In the present study, we used conditioned place preference (CPP) to assess the acquisition and maintenance of morphine conditioned place preference memory. The hippocampal protein level of RACK1 and synaptic quantitation were evaluated by Western blotting, immunohistochemistry and electron microscopy, respectively. Additionally, shRACK1 (shGnb2l1) was used to silence RACK1 in vivo to evaluate the role and the underlying mechanism of RACK1 in maintenance of morphine CPP memory. We found that morphine induced CPP was maintained for at least 7 days after the last morphine treatment, which indicated a positive correlation with hippocampal RACK1 level, and was accompanied simultaneously by increases in the synapse density and hippocampal expression of synaptophysin (SYP), phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2) and the phosphorylation of cyclic adenosine monophosphate response element-binding (pCREB). ShGnb2l1 icv injection significantly suppressed the expression of all above proteins, decreased the synapse density in the hippocampus and attenuated the acquisition and maintenance of morphine CPP. Our present study highlights that RACK1 plays an important role in the maintenance of morphine CPP, likely via activation of ERK-CREB pathway in hippocampus.

  1. Phosphorylation of Daxx by ATM Contributes to DNA Damage-Induced p53 Activation

    PubMed Central

    Cheng, Qian; Qu, Like; Brewer, Michael D.; Chen, Jiandong; Yang, Xiaolu

    2013-01-01

    p53 plays a central role in tumor suppression. It does so by inducing anti-proliferative processes as a response to various tumor-promoting stresses. p53 is regulated by the ubiquitin ligase Mdm2. The optimal function of Mdm2 requires Daxx, which stabilizes Mdm2 through the deubiquitinase Hausp/USP7 and also directly promotes Mdm2’s ubiquitin ligase activity towards p53. The Daxx-Mdm2 interaction is disrupted upon DNA damage. However, both the mechanisms and the consequence of the Daxx-Mdm2 dissociation are not understood. Here we show that upon DNA damage Daxx is phosphorylated in a manner that is dependent on ATM, a member of the PI 3-kinase family that orchestrates the DNA damage response. The main phosphorylation site of Daxx is identified to be Ser564, which is a direct target of ATM. Phosphorylation of endogenous Daxx at Ser564 occurs rapidly during the DNA damage response and precedes p53 activation. Blockage of this phosphorylation event prevents the separation of Daxx from Mdm2, stabilizes Mdm2, and inhibits DNA damage-induced p53 activation. These results suggest that phosphorylation of Daxx by ATM upon DNA damage disrupts the Daxx-Mdm2 interaction and facilitates p53 activation. PMID:23405218

  2. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    SciTech Connect

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-05-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of (/sup 32/P)-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions.

  3. Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L.

    PubMed Central

    Palmer, J. M.; Short, T. W.; Gallagher, S.; Briggs, W. R.

    1993-01-01

    Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity. PMID:12231896

  4. Aurora B is required for mitotic chromatin-induced phosphorylation of Op18/Stathmin.

    PubMed

    Gadea, Bedrick B; Ruderman, Joan V

    2006-03-21

    Oncoprotein 18/Stathmin (Op18) is a microtubule-destabilizing protein that is inhibited by phosphorylation in response to many types of signals. During mitosis, phosphorylation of Op18 by cdc2 is necessary but not sufficient for Op18 inhibition. The presence of mitotic chromosomes is additionally required and involves phosphorylation of Ser-16 in Xenopus Op18 (and/or Ser-63 in human). Given that Ser-16 is an excellent Aurora A (Aur-A) kinase consensus phosphorylation site and the Aurora kinase inhibitor ZM447439 (ZM) blocks phosphorylation in the activation loop of Aur-A, we asked whether either Aur-A or Aurora B (Aur-B) might regulate Op18. We find that ZM blocks the ability of mitotic chromatin to induce Op18 hyperphosphorylation in Xenopus egg extracts. Depletion of Aur-B, but not Aur-A, blocks hyperphosphorylation of Op18, and chromatin assembled in the absence of Aur-B fails to induce hyperphosphorylation. These results suggest that Aur-B, which concentrates at centromeres of metaphase chromosomes, contributes to localized regulation of Op18 during the process of spindle assembly.

  5. NALP1 is a transcriptional target for cAMP-response-element-binding protein (CREB) in myeloid leukaemia cells

    PubMed Central

    2004-01-01

    NALP1 (also called DEFCAP, NAC, CARD7) has been shown to play a central role in the activation of inflammatory caspases and processing of pro-IL1β (pro-interleukin-1β). Previous studies showed that NALP1 is highly expressed in peripheral blood mononuclear cells. In the present study, we report that expression of NALP1 is absent from CD34+ haematopoietic blast cells, and its levels are upregulated upon differentiation of CD34+ cells into granulocytes and to a lesser extent into monocytes. In peripheral blood cells, the highest levels of NALP1 were observed in CD3+ (T-lymphocytes), CD15+ (granulocytes) and CD14+ (monocytes) cell populations. Notably, the expression of NALP1 was significantly increased in the bone marrow blast cell population of some patients with acute leukaemia, but not among tissue samples from thyroid and renal cancer. A search for consensus sites within the NALP1 promoter revealed a sequence for CREB (cAMP-response-element-binding protein) that was required for transcriptional activity. Moreover, treatment of TF1 myeloid leukaemia cells with protein kinase C and protein kinase A activators induced CREB phosphorylation and upregulated the mRNA and protein levels of NALP1. Conversely, ectopic expression of a dominant negative form of CREB in TF1 cells blocked the transcriptional activity of the NALP1 promoter and significantly reduced the expression of NALP1. Thus NALP1 is transcriptionally regulated by CREB in myeloid cells, a mechanism that may contribute to modulate the response of these cells to pro-inflammatory stimuli. PMID:15285719

  6. A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

    PubMed Central

    Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei; Guikema, Jeroen E. J.; Schrader, Carol E.; Strauss, Phyllis R.

    2011-01-01

    DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex+/− mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes. PMID:21172930

  7. DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of {beta}-tubulin subtypes in the CNS of hens contributes to OPIDN

    SciTech Connect

    Damodaran, Tirupapuliyur V.; Gupta, Ram P.; Attia, Moustafa K.; Abou-Donia, Mohamed B.

    2009-10-15

    Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken, which results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 mL/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were sacrificed at different time points such as 2, 4, and 8 h, as well as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum brainstem and spinal cord were quickly dissected and frozen for protein (western) and mRNA (northern) studies. Subcellular fractionation, SDS-PAGE and immunoblotting of the nuclear and supernatant fractions using standard protocols from spinal cord and cerebrum showed differential expression of protein levels of PKA, CREB and phosphorylated CREB (p-CREB). There was an increase in PKA level in spinal cord nuclear fraction after 4 h (130 {+-} 5%) and 8 h (133 {+-} 6 %), while cerebrum nuclear fraction showed decrease (77 {+-} 5%) at 4 h and remained at the same level at 8 h. No change was seen in either spinal cord or cerebrum soluble fraction at any time points. There was an increase in CREB level in the spinal cord supernatant (133 {+-} 3%) after 5 days, while nuclear and supernatant fraction of the cerebrum did not show any alterations at any time point. p-CREB was induced in the spinal cord nuclear fraction at 1 day (150 {+-} 3%) and 5 days (173{+-}{+-}7%) of treatment, in contrast to the decreased levels p-CREB (72 {+-} 4%) at 10 days in cerebrum nuclear fraction. Supernatant fraction of spinal cord and cerebrum did

  8. Cardiac-specific overexpression of dominant-negative CREB leads to increased mortality and mitochondrial dysfunction in female mice.

    PubMed

    Watson, Peter A; Birdsey, Nicholas; Huggins, Gordon S; Svensson, Eric; Heppe, Daniel; Knaub, Leslie

    2010-12-01

    Cardiac failure is associated with diminished activation of the transcription factor cyclic nucleotide regulatory element binding-protein (CREB), and heart-specific expression of a phosphorylation-deficient CREB mutant in transgenic mice [dominant negative CREB (dnCREB) mice] recapitulates the contractile phenotypes of cardiac failure (Fentzke RC, Korcarz CE, Lang RM, Lin H, Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J Clin Invest 101: 2415-2426, 1998). In the present study, we demonstrated significantly elevated mortality and contractile dysfunction in female compared with male dnCREB mice. Female dnCREB mice demonstrated a 21-wk survival of only 17% compared with 67% in males (P < 0.05) and exclusively manifest decreased cardiac peroxisome proliferator-activated receptor-γ coactivator-1α and estrogen-related receptor-α content, suggesting sex-related effects on cardiac mitochondrial function. Hearts from 4-wk-old dnCREB mice of both sexes demonstrated diminished mitochondrial respiratory capacity compared with nontransgenic controls. However, by 12 wk of age, there was a significant decrease in mitochondrial density (citrate synthase activity) and deterioration of mitochondrial structure, as demonstrated by transmission electron microscopy, in female dnCREB mice, which were not found in male transgenic littermates. Subsarcolemmal mitochondria isolated from hearts of female, but not male, dnCREB mice demonstrated increased ROS accompanied by decreases in the expression/activity of the mitochondrial antioxidants MnSOD and glutathione peroxidase. These results demonstrate that heart-specific dnCREB expression results in mitochondrial respiratory dysfunction in both sexes; however, increased oxidant burden, reduced antioxidant expression, and disrupted mitochondrial structure are exacerbated by the female sex, preceding and contributing to the greater contractile morbidity and

  9. Tau phosphorylation and cleavage in ethanol-induced neurodegeneration in the developing mouse brain.

    PubMed

    Saito, Mariko; Chakraborty, Goutam; Mao, Rui-Fen; Paik, Sun-Mee; Vadasz, Csaba; Saito, Mitsuo

    2010-04-01

    Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3beta (GSK-3beta) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3beta and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3beta inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3beta and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.

  10. Extrasynaptic NMDA receptor-induced tau overexpression mediates neuronal death through suppressing survival signaling ERK phosphorylation

    PubMed Central

    Sun, Xu-Ying; Tuo, Qing-Zhang; Liuyang, Zhen-Yu; Xie, Ao-Ji; Feng, Xiao-Long; Yan, Xiong; Qiu, Mei; Li, Shen; Wang, Xiu-Lian; Cao, Fu-Yuan; Wang, Xiao-Chuan; Wang, Jian-Zhi; Liu, Rong

    2016-01-01

    Intracellular accumulation of the hyperphosphorylated tau is a pathological hallmark in the brain of Alzheimer disease. Activation of extrasynaptic NMDA receptors (E-NMDARs) induces excitatory toxicity that is involved in Alzheimer's neurodegeneration. However, the intrinsic link between E-NMDARs and the tau-induced neuronal damage remains elusive. In the present study, we showed in cultured primary cortical neurons that activation of E-NMDA receptors but not synaptic NMDA receptors dramatically increased tau mRNA and protein levels, with a simultaneous neuronal degeneration and decreased neuronal survival. Memantine, a selective antagonist of E-NMDARs, reversed E-NMDARs-induced tau overexpression. Activation of E-NMDARs in wild-type mouse brains resulted in neuron loss in hippocampus, whereas tau deletion in neuronal cultures and in the mouse brains rescued the E-NMDARs-induced neuronal death and degeneration. The E-NMDARs-induced tau overexpression was correlated with a reduced ERK phosphorylation, whereas the increased MEK activity, decreased binding and activity of ERK phosphatase to ERK, and increased ERK phosphorylation were observed in tau knockout mice. On the contrary, addition of tau proteins promoted ERK dephosphorylation in vitro. Taking together, these results indicate that tau overexpression mediates the excitatory toxicity induced by E-NMDAR activation through inhibiting ERK phosphorylation. PMID:27809304

  11. Oxidative stress–dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration

    PubMed Central

    Wakatsuki, Shuji; Furuno, Akiko; Ohshima, Makiko

    2015-01-01

    Oxidative stress is a well-known inducer of neuronal apoptosis and axonal degeneration. We previously showed that the E3 ubiquitin ligase ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B activation. We now demonstrate that oxidative stress serves as an activator of the ubiquitin ligase activity of ZNRF1 by inducing epidermal growth factor receptor (EGFR)–mediated phosphorylation at the 103rd tyrosine residue and that the up-regulation of ZNRF1 activity by oxidative stress leads to neuronal apoptosis and Wallerian degeneration. We also show that nicotinamide adenine dinucleotide phosphate–reduced oxidase activity is required for the EGFR-dependent phosphorylation-induced activation of ZNRF1 and resultant AKT degradation via the ubiquitin proteasome system to induce Wallerian degeneration. These results indicate the pathophysiological significance of the EGFR–ZNRF1 pathway induced by oxidative stress in the regulation of neuronal apoptosis and Wallerian degeneration. A deeper understanding of the regulatory mechanism for ZNRF1 catalytic activity via phosphorylation will provide a potential therapeutic avenue for neurodegeneration. PMID:26572622

  12. Hedgehog induces formation of PKA-Smoothened complexes to promote Smoothened phosphorylation and pathway activation

    PubMed Central

    Li, Shuang; Ma, Guoqiang; Wang, Bing; Jiang, Jin

    2015-01-01

    Hedgehog (Hh) is a secreted glycoprotein that binds its receptor Patched to activate the G protein-coupled receptor-like protein Smoothened (Smo). In Drosophila, protein kinase A (PKA) phosphorylates and activates Smo in cells stimulated with Hh. In unstimulated cells, PKA phosphorylates and inhibits the transcription factor Cubitus interruptus (Ci). Here, we found that in cells exposed to Hh, the catalytic subunit of PKA (PKAc) bound to the juxtamembrane region of the C terminus of Smo. PKA-mediated phosphorylation of Smo further enhanced its association with PKAc to form stable kinase-substrate complexes that promoted the PKA-mediated trans-phosphorylation of Smo dimers. We identified multiple basic residues in the C-terminus of Smo that were required for interaction with PKAc, Smo phosphorylation, and Hh pathway activation. Hh induced a switch from the association of PKAc with a cytosolic complex of Ci and the kinesin-like protein Costal2 (Cos2) to a membrane-bound Smo-Cos2 complex. Thus, our study uncovers a previously uncharacterized mechanism for regulation of PKA activity and demonstrates that the signal-regulated formation of kinase-substrate complexes plays a central role in Hh signal transduction. PMID:24985345

  13. Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor.

    PubMed

    Flores, Rosa V; Hernández-Pérez, Melvin G; Aquino, Edna; Garrad, Richard C; Weisman, Gary A; Gonzalez, Fernando A

    2005-12-01

    Purification of HA-tagged P2Y2 receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57-76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y2 receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y2 nucleotide receptors from metabolically [32P]-labelled cells indicated a (3.8 +/- 0.2)-fold increase in the [32P]-content of the receptor after 15 min of treatment with 100 microM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that approximately 40% of the surface receptors were internalized after a 15-min stimulation with 100 microM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y2 receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y2 receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y2 receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y2 nucleotide receptor.

  14. Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

    PubMed

    Corbett, Grant T; Roy, Avik; Pahan, Kalipada

    2013-03-22

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

  15. Lysophosphatidic Acid Induces Neurite Retraction in Differentiated Neuroblastoma Cells via GSK-3β Activation

    PubMed Central

    Sun, Yuanjie; Kim, Nam-Ho; Yang, Haijie; Kim, Seung-Hyuk; Huh, Sung-Oh

    2011-01-01

    Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects, including rapid neurite retraction and cell migration. Alterations in cell morphology, including neurite retraction, in neurodegenerative disorders such as Alzheimer’s disease involve hyperphosphorylation of the cytoskeletal protein tau. Since LPA has been shown to induce neurite retraction in various cultured neural cells and the detailed underlying molecular mechanisms have not yet been elucidated, we investigated whether LPA induced neurite retraction through taumediated signaling pathways in differentiated neuroblastoma cells. When Neuro2a cells differentiated with retinoic acid (RA) were exposed to LPA, cells exhibited neurite retraction in a time-dependent manner. The retraction of neurites was accompanied by the phosphorylation of tau. The LPA-induced neurite retraction and tau phosphorylation in differentiated Neuro2a cells were significantly abolished by the glycogen synthase kinase-3β (GSK-3β) inhibitor lithium chloride. Interestingly, the LPA-stimulated tau phosphorylation and neurite retraction were markedly prevented by the administration of H89, an inhibitor of both cyclic-AMP dependent protein kinase (PKA) and cyclic- AMP response element-binding protein (CREB). Transfection of the dominant-negative CREBs, K-CREB and ACREB, failed to prevent LPA-induced tau phosphorylation and neurite retraction in differentiated Neuro2a cells. Taken together, these results suggest that GSK-3β and PKA, rather than CREB, play important roles in tau phosphorylation and neurite retraction in LPA-stimulated differentiated Neuro2a cells. PMID:21499833

  16. Microglia activation regulates GluR1 phosphorylation in chronic unpredictable stress-induced cognitive dysfunction.

    PubMed

    Liu, Mingchao; Li, Juan; Dai, Peng; Zhao, Fang; Zheng, Gang; Jing, Jinfei; Wang, Jiye; Luo, Wenjing; Chen, Jingyuan

    2015-01-01

    Chronic stress is considered to be a major risk factor in the development of psychopathological syndromes in humans. Cognitive impairments and long-term potentiation (LTP) impairments are increasingly recognized as major components of depression, anxiety disorders and other stress-related chronic psychological illnesses. It seems timely to systematically study the potentially underlying neurobiological mechanisms of altered cognitive and synaptic plasticity in the course of chronic stress. In the present study, a rat model of chronic unpredictable stress (CUS) induced a cognitive impairment in spatial memory in the Morris water maze (MWM) test and a hippocampal LTP impairment. CUS also induced hippocampal microglial activation and attenuated phosphorylation of glutamate receptor 1 (GluR1 or GluA1). Moreover, chronic treatment with the selective microglial activation blocker, minocycline (120 mg/kg per day), beginning 3 d before CUS treatment and continuing through the behavioral testing period, prevented the CUS-induced impairments of spatial memory and LTP induction. Additional studies showed that minocycline-induced inhibition of microglia activation was associated with increased phosphorylation of GluR1. These results suggest that hippocampal microglial activation modulates the level of GluR1 phosphorylation and might play a causal role in CUS-induced cognitive and LTP disturbances.

  17. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    SciTech Connect

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  18. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    PubMed

    Goel, Anubhuti; Xu, Linda W; Snyder, Kevin P; Song, Lihua; Goenaga-Vazquez, Yamila; Megill, Andrea; Takamiya, Kogo; Huganir, Richard L; Lee, Hey-Kyoung

    2011-03-31

    Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  19. Intracerebroventricular administration of okadaic acid induces hippocampal glucose uptake dysfunction and tau phosphorylation.

    PubMed

    Broetto, Núbia; Hansen, Fernanda; Brolese, Giovana; Batassini, Cristiane; Lirio, Franciane; Galland, Fabiana; Dos Santos, João Paulo Almeida; Dutra, Márcio Ferreira; Gonçalves, Carlos-Alberto

    2016-06-01

    Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimer's disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism.

  20. Regulatory light chain phosphorylation increases eccentric contraction-induced injury in skinned fast-twitch fibers.

    PubMed

    Childers, Martin K; McDonald, Kerry S

    2004-02-01

    During contraction, activation of Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) results in phosphorylation of myosin's regulatory light chain (RLC), which potentiates force and increases speed of force development over a wide range of [Ca(2+)]. We tested the hypothesis that RLC phosphorylation by MLCK mediates the extent of eccentric contraction-induced injury as measured by force deficit in skinned fast-twitch skeletal muscle fibers. Results indicated that RLC phosphorylation in single skinned rat psoas fibers significantly increased Ca(2+) sensitivity of isometric force; isometric force from 50 +/- 16 to 59 +/- 18 kN/m(2) during maximal Ca(2+) activation; peak absolute power output from 38 +/- 15 to 48 +/- 14 nW during maximal Ca(2+) activation; and the magnitude of contraction-induced force deficit during maximal (pCa 4.5) activation from 26 +/- 9.8 to 35 +/- 9.6%. We conclude that RLC phosphorylation increases force deficits following eccentric contractions, perhaps by increasing the number of force-generating cross-bridges.

  1. Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection.

    PubMed

    Musicki, Biljana; Palese, Michael A; Crone, Julie K; Burnett, Arthur L

    2004-02-01

    The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.

  2. UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53{sup Ser-15} phosphorylation

    SciTech Connect

    Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader; Shahin, Allen; Patel, Mahendra; Galadari, Sehamuddin

    2009-06-12

    Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser{sup 15}. Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not prevent Dubca cell apoptosis, suggesting that p53{sup Ser-15} phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.

  3. Mitochondrial dysfunction induces dendritic loss via eIF2α phosphorylation

    PubMed Central

    Tsuyama, Taiichi; Tsubouchi, Asako; Imamura, Hiromi

    2017-01-01

    Mitochondria are key contributors to the etiology of diseases associated with neuromuscular defects or neurodegeneration. How changes in cellular metabolism specifically impact neuronal intracellular processes and cause neuropathological events is still unclear. We here dissect the molecular mechanism by which mitochondrial dysfunction induced by Prel aberrant function mediates selective dendritic loss in Drosophila melanogaster class IV dendritic arborization neurons. Using in vivo ATP imaging, we found that neuronal cellular ATP levels during development are not correlated with the progression of dendritic loss. We searched for mitochondrial stress signaling pathways that induce dendritic loss and found that mitochondrial dysfunction is associated with increased eIF2α phosphorylation, which is sufficient to induce dendritic pathology in class IV arborization neurons. We also observed that eIF2α phosphorylation mediates dendritic loss when mitochondrial dysfunction results from other genetic perturbations. Furthermore, mitochondrial dysfunction induces translation repression in class IV neurons in an eIF2α phosphorylation-dependent manner, suggesting that differential translation attenuation among neuron subtypes is a determinant of preferential vulnerability. PMID:28209644

  4. Human TNF-α induces differential protein phosphorylation in Schistosoma mansoni adult male worms.

    PubMed

    Oliveira, Katia C; Carvalho, Mariana L P; Bonatto, José Matheus C; Schechtman, Debora; Verjovski-Almeida, Sergio

    2016-02-01

    Schistosoma mansoni and its vertebrate host have a complex and intimate connection in which several molecular stimuli are exchanged and affect both organisms. Human tumor necrosis factor alpha (hTNF-α), a pro-inflammatory cytokine, is known to induce large-scale gene expression changes in the parasite and to affect several parasite biological processes such as metabolism, egg laying, and worm development. Until now, the molecular mechanisms for TNF-α activity in worms are not completely understood. Here, we aimed at exploring the effect of hTNF-α on S. mansoni protein phosphorylation by 2D gel electrophoresis followed by a quantitative analysis of phosphoprotein staining and protein identification by mass spectrometry. We analyzed three biological replicates of adult male worms exposed to hTNF-α and successfully identified 32 protein spots with a statistically significant increase in phosphorylation upon in vitro exposure to hTNF-α. Among the differentially phosphorylated proteins, we found proteins involved in metabolism, such as glycolysis, galactose metabolism, urea cycle, and aldehyde metabolism, as well as proteins related to muscle contraction and to cytoskeleton remodeling. The most differentially phosphorylated protein (30-fold increase in phosphorylation) was 14-3-3, whose function is known to be modulated by phosphorylation, belonging to a signal transduction protein family that regulates a variety of processes in all eukaryotic cells. Further, 75% of the identified proteins are known in mammals to be related to TNF-α signaling, thus suggesting that TNF-α response may be conserved in the parasite. We propose that this work opens new perspectives to be explored in the study of the molecular crosstalk between host and pathogen.

  5. Hippocampal overexpression of mutant creb blocks long-term, but not short-term memory for a socially transmitted food preference.

    PubMed

    Brightwell, Jennifer J; Smith, Clayton A; Countryman, Renee A; Neve, Rachael L; Colombo, Paul J

    2005-01-01

    Phosphorylation of the transcription factor CREB on Ser133 is implicated in the establishment of long-term memory for hippocampus-dependent tasks, including spatial learning and contextual fear conditioning. We reported previously that training on a hippocampus-dependent social transmission of food preference (STFP) task increases CREB phosphorylation in the hippocampus of trained rats in comparisons with controls. In the current study, we tested the hypothesis that CREB function is necessary for long-term memory for STFP using herpes simplex viral (HSV) vector-mediated gene transfer. Rats received intrahippocampal infusions of HSV-mCREB (a mutant form of CREB, in which Ser133 has been replaced with Ala), HSV-LacZ, or saline, and were trained 3 d later. Rats were tested for food preference (demonstrated vs. novel foods) immediately (short-term test) and 11 d (long-term test) after training. Rats in all treatment groups showed a significant preference for the demonstrated food at the short-term memory test. At the long-term memory test, however, the percentage of demonstrated food eaten by mCREB-treated rats was significantly less than that eaten by the LacZ- or saline-treated rats. Quantitative Western blotting confirmed that mCREB-infused rats had significantly more hippocampal CREB protein than controls during training. The present results show that hippocampal CREB function is necessary for long-term, but not short-term memory for STFP.

  6. Conventional protein kinase C isoforms mediate phorbol ester-induced lysophosphatidic acid LPA1 receptor phosphorylation.

    PubMed

    Hernández-Méndez, Aurelio; Alcántara-Hernández, Rocío; Acosta-Cervantes, Germán C; Martínez-Ortiz, Javier; Avendaño-Vázquez, S Eréndira; García-Sáinz, J Adolfo

    2014-01-15

    Using C9 cells stably expressing LPA1 receptors fused to the enhanced green fluorescent protein, it was observed that activation of protein kinase C induced a rapid and strong increase in the phosphorylation state of these receptors. Overnight incubation with phorbol esters markedly decreased the amount of conventional (α, βI, βII and γ) and novel (δ) but not atypical (ζ) immunodetected PKC isoforms, this treatment blocks the action of protein kinase on receptor function and phosphorylation. Bis-indolylmaleimide I a general, non-subtype selective protein kinase C inhibitor, and Gö 6976, selective for the isoforms α and β, were also able to block LPA1 receptor desensitization and phosphorylation; hispidin, isoform β-selective blocker partially avoided receptor desensitization. Expression of dominant-negative protein kinase C α or β II mutants and knocking down the expression of these kinase isozymes markedly decreased phorbol ester-induced LPA1 receptor phosphorylation without avoiding receptor desensitization. This effect was blocked by bis-indolyl-maleimide and Gö 6976, suggesting that these genetic interventions were not completely effective. It was also observed that protein kinase C α and β II isozymes co-immunoprecipitate with LPA1 receptors and that such an association was further increased by cell treatments with phorbol esters or lysophosphatidic acid. Our data suggest that conventional protein kinase C α and β isozymes modulate LPA1 receptor phosphorylation state. Receptor desensitization appears to be a more complex process that might involve additional elements. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Ganglioside GD3 monoclonal antibody-induced paxillin tyrosine phosphorylation and filamentous actin assembly in cerebellar growth cones.

    PubMed

    Yuyama, Kohei; Sekino-Suzuki, Naoko; Yamamoto, Naomasa; Kasahara, Kohji

    2011-03-01

    We have demonstrated that antibody to ganglioside GD3 (R24) immunoprecipitates src-family tyrosine kinase Lyn from primary cerebellar granule cells and R24 treatment of the intact cells induces Lyn activation and rapid tyrosine phosphorylation of several substrates, suggesting the functional association of ganglioside GD3 with Lyn. In this study, R24 treatment of primary cerebellar granule cells enhances phosphorylation of paxillin at tyrosine residue 118 and induces filamentous actin assembly and neurite outgrowth. R24 treatment of cerebellar growth cone membrane fraction induces prominent tyrosine phosphorylation of 68 kDa protein which comigrates with phosphopaxillin at tyrosine residue 118. Tyrosine phosphorylation of paxillin is known to regulate actin cytoskeleton-dependent changes in cell morphology. Signal transduction by ganglioside GD3 is involved in growth cone morphology via tyrosine phosphorylation of paxillin. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  8. Primary Blast-Induced Changes in Akt and GSK3β Phosphorylation in Rat Hippocampus

    PubMed Central

    Wang, Yushan; Sawyer, Thomas W.; Tse, Yiu Chung; Fan, Changyang; Hennes, Grant; Barnes, Julia; Josey, Tyson; Weiss, Tracy; Nelson, Peggy; Wong, Tak Pan

    2017-01-01

    Traumatic brain injury (TBI) due to blast from improvised explosive devices has been a leading cause of morbidity and mortality in recent conflicts in Iraq and Afghanistan. However, the mechanisms of primary blast-induced TBI are not well understood. The Akt signal transduction pathway has been implicated in various brain pathologies including TBI. In the present study, the effects of simulated primary blast waves on the phosphorylation status of Akt and its downstream effector kinase, glycogen synthase kinase 3β (GSK3β), in rat hippocampus, were investigated. Male Sprague-Dawley (SD) rats (350–400 g) were exposed to a single pulse shock wave (25 psi; ~7 ms duration) and sacrificed 1 day, 1 week, or 6 weeks after exposure. Total and phosphorylated Akt, as well as phosphorylation of its downstream effector kinase GSK3β (at serine 9), were detected with western blot analysis and immunohistochemistry. Results showed that Akt phosphorylation at both serine 473 and threonine 308 was increased 1 day after blast on the ipsilateral side of the hippocampus, and this elevation persisted until at least 6 weeks postexposure. Similarly, phosphorylation of GSK3β at serine 9, which inhibits GSK3β activity, was also increased starting at 1 day and persisted until at least 6 weeks after primary blast on the ipsilateral side. In contrast, p-Akt was increased at 1 and 6 weeks on the contralateral side, while p-GSK3β was increased 1 day and 1 week after primary blast exposure. No significant changes in total protein levels of Akt and GSK were observed on either side of the hippocampus at any time points. Immunohistochemical results showed that increased p-Akt was mainly of neuronal origin in the CA1 region of the hippocampus and once phosphorylated, the majority was translocated to the dendritic and plasma membranes. Finally, electrophysiological data showed that evoked synaptic N-methyl-d-aspartate (NMDA) receptor activity was significantly increased

  9. GSK3beta-mediated Drp1 phosphorylation induced elongated mitochondrial morphology against oxidative stress.

    PubMed

    Chou, Chia-Hua; Lin, Ching-Chih; Yang, Ming-Chang; Wei, Chih-Chang; Liao, Huei-De; Lin, Run-Chin; Tu, Wen-Yu; Kao, Tsung-Chieh; Hsu, Ching-Mei; Cheng, Jiin-Tsuey; Chou, An-Kuo; Lee, Chu-I; Loh, Joon-Khim; Howng, Shen-Long; Hong, Yi-Ren

    2012-01-01

    Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634~736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H(2)O(2)-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H(2)O(2) insult.

  10. GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress

    PubMed Central

    Wei, Chih-Chang; Liao, Huei-De; Lin, Run-Chin; Tu, Wen-Yu; Kao, Tsung-Chieh; Hsu, Ching-Mei; Cheng, Jiin-Tsuey; Chou, An-Kuo; Lee, Chu-I; Loh, Joon-Khim; Howng, Shen-Long; Hong, Yi-Ren

    2012-01-01

    Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634∼736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H2O2-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H2O2 insult. PMID:23185298

  11. A Large-Scale Quantitative Proteomic Approach To Identifying Sulfur Mustard-Induced Protein Phosphorylation Cascades

    DTIC Science & Technology

    2009-07-31

    are no effective treatments for SM-induced injury, current research focuses on understanding the molecular changes upon SM exposure. Indeed, efforts...with immobilized metal affinity chromatography to study the large-scale protein phosphorylation changes resulting from SM exposure in a human... effective at probing individual pathways, they do not put into context the global changes that are occurring in response to SM and how these many

  12. EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    PubMed Central

    Chun, Kyung-Soo; Shim, Minsub

    2015-01-01

    Prostaglandin E2 (PGE2), a major product of cyclooxygenase, binds to four different prostaglandin E2 receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of PGE2 induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and β-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway. PMID:26535079

  13. Advanced glycation end products induce moesin phosphorylation in murine retinal endothelium.

    PubMed

    Wang, Lingjun; Li, Qiaoqin; Du, Jing; Chen, Bo; Li, Qiang; Huang, Xuliang; Guo, Xiaohua; Huang, Qiaobing

    2012-02-01

    Increase in vascular permeability is the most important pathological event during the development of diabetic retinopathy. Deposition of advanced glycation end products (AGEs) plays a crucial role in the process of diabetes. This study was to investigate the role of moesin and its underlying signal transduction in retinal vascular hyper-permeability induced by AGE-modified mouse serum albumin (AGE-MSA). Female C57BL/6 mice were used to produce an AGE-treated model by intraperitoneal administration of AGE-MSA for seven consecutive days. The inner blood-retinal barrier was quantified by Evans blue leakage assay. Endothelial F-actin cytoskeleton in retinal vasculature was visualized by fluorescence probe staining. The expression and phosphorylation of moesin in retinal vessels were detected by RT-PCR and western blotting. Further studies were performed to explore the effects of Rho kinase (ROCK) and p38 MAPK pathway on the involvement of moesin in AGE-induced retinal vascular hyper-permeability response. Treatment with AGE-MSA significantly increased the permeability of the retinal microvessels and induced the disorganization of F-actin in retinal vascular endothelial cells. The threonine (T558) phosphorylation of moesin in retinal vessels was enhanced remarkably after AGE administration. The phosphorylation of moesin was attenuated by inhibitions of ROCK and p38 MAPK, while this treatment also prevented the dysfunction of inner blood-retinal barrier and the reorganization of F-actin in retinal vascular endothelial cells. These results demonstrate that moesin is involved in AGE-induced retinal vascular endothelial dysfunction and the phosphorylation of moesin is triggered via ROCK and p38 MAPK activation.

  14. Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

    SciTech Connect

    Geiss, Brian J.; Cano, Gina L.; Tavis, John E.; Morrison, Lynda A. . E-mail: morrisla@slu.edu

    2004-12-05

    Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

  15. Inducible enhancement of memory storage and synaptic plasticity in transgenic mice expressing an inhibitor of ATF4 (CREB-2) and C/EBP proteins.

    PubMed

    Chen, Amy; Muzzio, Isabel A; Malleret, Gaël; Bartsch, Dusan; Verbitsky, Miguel; Pavlidis, Paul; Yonan, Amanda L; Vronskaya, Svetlana; Grody, Michael B; Cepeda, Ivan; Gilliam, T Conrad; Kandel, Eric R

    2003-08-14

    To examine the role of C/EBP-related transcription factors in long-term synaptic plasticity and memory storage, we have used the tetracycline-regulated system and expressed in the forebrain of mice a broad dominant-negative inhibitor of C/EBP (EGFP-AZIP), which preferentially interacts with several inhibiting isoforms of C/EBP. EGFP-AZIP also reduces the expression of ATF4, a distant member of the C/EBP family of transcription factors that is homologous to the Aplysia memory suppressor gene ApCREB-2. Consistent with the removal of inhibitory constraints on transcription, we find an increase in the pattern of gene transcripts in the hippocampus of EGFP-AZIP transgenic mice and both a reversibly enhanced hippocampal-based spatial memory and LTP. These results suggest that several proteins within the C/EBP family including ATF4 (CREB-2) act to constrain long-term synaptic changes and memory formation. Relief of this inhibition lowers the threshold for hippocampal-dependent long-term synaptic potentiation and memory storage in mice.

  16. AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair

    PubMed Central

    Jarrett, Stuart G.; Wolf Horrell, Erin M.; D'Orazio, John A.

    2016-01-01

    Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12–ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5′ strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12–ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER. PMID:27683220

  17. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    SciTech Connect

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  18. CDK5 phosphorylates DRP1 and drives mitochondrial defects in NMDA-induced neuronal death.

    PubMed

    Jahani-Asl, Arezu; Huang, En; Irrcher, Isabella; Rashidian, Juliet; Ishihara, Naotada; Lagace, Diane C; Slack, Ruth S; Park, David S

    2015-08-15

    Defects in mitochondrial fission and cyclin dependent kinase 5 (CDK5) activation are early events that precede neuronal loss following NMDA-induced neuronal death. Here, we report that the cytoplasmic CDK5 tightly regulates mitochondrial morphology defects associated with NMDA-induced neuronal injury via regulation of the mitochondrial fission protein, dynamin-related protein 1 (DRP1). We show that DRP1 is a direct target of CDK5. CDK5-mediated phosphorylation of DRP1 at a conserved Serine residue, S585, is elevated at the mitochondria and is associated with increased mitochondrial fission. Ectopic expression of a cytoplasmic CDK5 or mutant DRP1-S585D results in increased mitochondrial fragmentation in primary neurons. Conversely, expression of a dominant negative form of cytoplasmic CDK5 or mutant DRP1-S585A results in elongated mitochondria. In addition, pharmacological inhibition of CDK5 by Roscovitine inhibits DRP1 phosphorylation and mitochondrial fission associated with NMDA-induced neuronal loss. Importantly, conditional deletion of CDK5 significantly attenuates DRP1 phosphorylation at S585 and rescues mitochondrial fission defects in neurons exposed to NMDA. Our studies delineate an important mechanism by which CDK5 regulates mitochondrial morphology defects associated with neuronal injury.

  19. TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

    PubMed Central

    Fusté, Berta; Díaz-Ricart, Maribel; Jensen, Morten Krogh; Ordinas, Antonio; Escolar, Ginés; White, James G.

    2002-01-01

    We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and α-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease. PMID:12057926

  20. Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy*

    PubMed Central

    Puente, Cindy; Hendrickson, Ronald C.; Jiang, Xuejun

    2016-01-01

    Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation. PMID:26801615

  1. Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy.

    PubMed

    Puente, Cindy; Hendrickson, Ronald C; Jiang, Xuejun

    2016-03-11

    Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.

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

    PubMed

    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.

  3. Auto-inhibition and phosphorylation-induced activation of PLC-γ isozymes

    PubMed Central

    Hajicek, Nicole; Charpentier, Thomas H.; Rush, Jeremy R.; Harden, T. Kendall; Sondek, John

    2013-01-01

    Multiple extracellular stimuli, such as growth factors and antigens, initiate signaling cascades through tyrosine phosphorylation and activation of phospholipase C (PLC)-γ isozymes. Like most other PLCs, PLC-γ1 is basally auto-inhibited by its X-Y linker, which separates the X-and Y-boxes of the catalytic core. The C-terminal SH2 (cSH2) domain within the X-Y linker is the critical determinant for auto-inhibition of phospholipase activity. Release of auto-inhibition requires an intramolecular interaction between the cSH2 domain and a phosphorylated tyrosine, Tyr783, also located within the X-Y linker. The molecular mechanisms that mediate auto-inhibition and phosphorylation-induced activation have not been defined. Here, we describe structures of the cSH2 domain both alone and bound to a PLC-γ1 peptide encompassing phosphorylated Tyr783. The cSH2 domain remains largely unaltered by peptide engagement. Point mutations in the cSH2 domain located at the interface with the peptide were sufficient to constitutively activate PLC-γ1 suggesting that peptide engagement directly interferes with the capacity of the cSH2 domain to block the lipase active site. This idea is supported by mutations in a complimentary surface of the catalytic core that also enhanced phospholipase activity. PMID:23777354

  4. Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

    PubMed

    Abushahba, Walid; Olabisi, Oyenike O; Jeong, Byeong-Seon; Boregowda, Rajeev K; Wen, Yu; Liu, Fang; Goydos, James S; Lasfar, Ahmed; Cohen-Solal, Karine A

    2012-01-01

    Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.

  5. Non-Canonical Smads Phosphorylation Induced by the Glutamate Release Inhibitor, Riluzole, through GSK3 Activation in Melanoma

    PubMed Central

    Jeong, Byeong-Seon; Boregowda, Rajeev K.; Wen, Yu; Liu, Fang; Goydos, James S.; Lasfar, Ahmed; Cohen-Solal, Karine A.

    2012-01-01

    Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas. PMID:23077590

  6. The effects of in vitro phosphorylation and dephosphorylation on the thrombin-induced gelation and plasmin degradation of fibrinogen.

    PubMed

    Martin, S C; Forsberg, P O; Eriksson, S D

    1991-02-01

    The alpha-chain of human fibrinogen was found to be phosphorylated in EDTA-anticoagulated whole blood when trace amounts of (gamma-32P)ATP and 7.5 mM Mg2+ ions were added. Fibrinogen was not phosphorylated if only the ATP was added. The thrombin-induced gelation of fibrinogen phosphorylated by protein kinase A, casein kinase I or II was studied spectrophotomerically. It was found that phosphorylation by protein kinase A caused the formation of thinner fibrin fibres, whereas phosphorylation by casein kinase II resulted in fibres slightly thicker than those of the control fibrinogen (equivalent to a 20% increase in the control fibrinogen concentration). Phosphorylation with casein kinase I did not significantly affect the fibrin fibre thickness. Dephosphorylation by alkaline phosphatase removed 50% of the 32P-labelled phosphate from protein kinase A-phosphorylated fibrinogen and over 90% from the casein kinase I or II-phosphorylated fibrinogens. This dephosphorylation resulted in a general increase in fibre thickness in the gelation assay in all samples, although the fibres of the phosphorylated fibrinogens remained substantially thinner than the dephosphorylated control fibrinogen. Plasmin digestion of the phosphorylated fibrinogens showed that they were more resistant to cleavage, being cleaved at only 30% to 70% of the rate of control fibrinogen and that this resistance was unaltered by dephosphorylation, in contrast to the thrombin gelation experiments.

  7. Genome-wide functional analysis of CREB/long-term memory-dependent transcription reveals distinct basal and memory gene expression programs.

    PubMed

    Lakhina, Vanisha; Arey, Rachel N; Kaletsky, Rachel; Kauffman, Amanda; Stein, Geneva; Keyes, William; Xu, Daniel; Murphy, Coleen T

    2015-01-21

    Induced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components.

  8. Genome-wide Functional Analysis of CREB/Long-Term Memory-Dependent Transcription Reveals Distinct Basal and Memory Gene Expression Programs

    PubMed Central

    Lakhina, Vanisha; Arey, Rachel N.; Kaletsky, Rachel; Kauffman, Amanda; Stein, Geneva; Keyes, William; Xu, Daniel; Murphy, Coleen T.

    2014-01-01

    SUMMARY Induced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components. PMID:25611510

  9. Calcium/calmodulin dependent protein kinase II regulates the phosphorylation of cyclic AMP-responsive element-binding protein of spinal cord in rats following noxious stimulation.

    PubMed

    Fang, Li; Wu, Jing; Zhang, Xuan; Lin, Qing; Willis, William D

    2005-02-01

    We have previously reported that intradermal capsaicin injection causes the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats. The present study was designed to investigate the role of calcium/camodulin protein dependent protein kinase II (CaM kinase II) in the regulation of phosphorylation of CREB after capsaicin injection. We found that capsaicin injection produces a significant upregulation of phosphorylated CREB in the spinal cord of rat. Intrathecal treatment with a CaM kinase II inhibitor, KN-93, significantly blocked the increased phosphorylation of CREB, but did not affect the CREB protein itself. These results suggest that increased phosphorylation of CREB protein may contribute to central sensitization following acute peripheral noxious stimuli, and the effect may be regulated through the activation of CaM kinase cascades.

  10. TAUROLITHOCHOLATE-INDUCED MRP2 RETRIEVAL INVOLVES MARCKS PHOSPHORYLATION BY PROTEIN KINASE Cε IN HUH-NTCP CELLS

    PubMed Central

    Schonhoff, Christopher M.; Webster, Cynthia R. L.; Anwer, M. Sawkat

    2013-01-01

    Taurolithocholate (TLC) acutely inhibits biliary excretion of Mrp2 substrates by inducing Mrp2 retrieval from the canalicular membrane, while cAMP increases plasma membrane MRP2. The effect of TLC may be mediated via protein kinase Cε (PKCε). Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is a membrane bound F-actin cross linking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis and the underlying mechanism appears to be detachment of phosphorylated MARCKS from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCε mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with NTCP (HuH-NTCP cells) and in rat hepatocytes. TLC increased plasma membrane (PM) PKCε and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. Cyclic AMP did not affect PM-PKCε and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant negative (DN) PKCε reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and PMA increased cytosolic phospho-MARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCε suggesting PKCε mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation deficient MARCKS, TLC failed to increase MARCKS phosphorylation and to decrease plasma membrane MRP2. Conclusion Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC mediated activation of PKCε followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane. PMID:23424156

  11. MTOR inhibition attenuates DNA damage and apoptosis through autophagy-mediated suppression of CREB1.

    PubMed

    Wang, Ying; Hu, Zhongdong; Liu, Zhibo; Chen, Rongrong; Peng, Haiyong; Guo, Jing; Chen, Xinxin; Zhang, Hongbing

    2013-12-01

    Hyperactivation of mechanistic target of rapamycin (MTOR) is a common feature of human cancers, and MTOR inhibitors, such as rapamycin, are thus becoming therapeutics in targeting certain cancers. However, rapamycin has also been found to compromise the efficacy of chemotherapeutics to cells with hyperactive MTOR. Here, we show that loss of TSC2 or PTEN enhanced etoposide-induced DNA damage and apoptosis, which was blunted by suppression of MTOR with either rapamycin or RNA interference. cAMP response element-binding protein 1 (CREB1), a nuclear transcription factor that regulates genes involved in survival and death, was positively regulated by MTOR in mouse embryonic fibroblasts (MEFs) and cancer cell lines. Silencing Creb1 expression with siRNA protected MTOR-hyperactive cells from DNA damage-induced apoptosis. Furthermore, loss of TSC2 or PTEN impaired either etoposide or nutrient starvation-induced autophagy, which in turn, leads to CREB1 hyperactivation. We further elucidated an inverse correlation between autophagy activity and CREB1 activity in the kidney tumor tissue obtained from a TSC patient and the mouse livers with hepatocyte-specific knockout of PTEN. CREB1 induced DNA damage and subsequent apoptosis in response to etoposide in autophagy-defective cells. Reactivation of CREB1 or inhibition of autophagy not only improved the efficacy of rapamycin but also alleviated MTOR inhibition-mediated chemoresistance. Therefore, autophagy suppression of CREB1 may underlie the MTOR inhibition-mediated chemoresistance. We suggest that inhibition of MTOR in combination with CREB1 activation may be used in the treatment of cancer caused by an abnormal PI3K-PTEN-AKT-TSC1/2-MTOR signaling pathway. CREB1 activators should potentiate the efficacy of chemotherapeutics in treatment of these cancers.

  12. [Escherichia coli L-asparaginase induces phosphorylation of endogenous polypeptides in human immune cells].

    PubMed

    Mercado, L; Arenas, G

    1999-12-01

    To detect patterns of endogenous polypeptide phosphorylation in monocyte, lymphocyte, and polymorphonuclear leukocyte populations, induced by the products of the catalytic action of L-asparaginase (EcA). Monocytes, polymorphonuclear cells and lymphocytes were isolated from heparinized blood from healthy, voluntary donors. The samples were incubated in 0.4 mCi/ml of [gamma-32P]H3PO4, with: 1 microgram/microliter of EcA, EcA and the substrate or with the products of EcA's catalytic activity: NH4+ and aspartate. The cells were lysated and electrophoresed using denaturing polyacrylamide gels that were then exposed on radiographic plates. The levels of polypeptide phosphorylation were quantified by computer densitometric analysis. The autoradiographs and the densitometric quantification of the electrophoretic profiles of monocytes, polymorphonuclear leukocytes, and lymphocytes revealed an increase in polypeptide phosphorylation when the cells were incubated with the enzyme and its substrate, ammonium and aspartate, or ammonium, which demonstrates that the NH4+ triggers intracellular phosphotransferase activity. A 58 kDa phosphoprotein outstood, it being common to the three cell populations studied. There were also specific phosphorylable polypeptides in monocytes, polymorphonuclear leukocytes, and lymphocytes. Escherichia coli L-asparaginase, binds the plasma membrane in normal human immune cells, catalyzing the L-asparagine substrate. The products of its activity: aspartate and NH4+ modify the extracellular environment, particularly the latter since it could diffuse into the cytosol and modify the pH, which would activate signal transduction pathways associated with the phosphorylation of substrates.

  13. Juvenile Hormone Prevents 20-Hydroxyecdysone-induced Metamorphosis by Regulating the Phosphorylation of a Newly Identified Broad Protein*

    PubMed Central

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-01-01

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

  14. Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

    PubMed

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-09-19

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

    PubMed

    Goldbart, A; Row, B W; Kheirandish, L; Schurr, A; Gozal, E; Guo, S Z; Payne, R S; Cheng, Z; Brittian, K R; Gozal, D

    2003-01-01

    Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

  16. Cadmium induces phosphorylation and stabilization of c-Fos in HK-2 renal proximal tubular cells

    SciTech Connect

    Iwatsuki, Mamiko; Inageda, Kiyoshi; Matsuoka, Masato

    2011-03-15

    We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the expression and phosphorylation status of members of the Fos family, components of the activator protein-1 transcription factor, in HK-2 human renal proximal tubular cells. Following the exposure to CdCl{sub 2}, the expression of c-fos, fosB, fra-1, and fra-2 increased markedly, with different magnitudes and time courses. The levels of Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) also increased in response to CdCl{sub 2} exposure. Although the elevation of c-fos transcripts was transient, c-Fos protein levels increased progressively with lower electrophoretic mobility, suggesting stabilization of c-Fos through post-translational modifications. Consistently, we observed phosphorylation of c-Fos at Ser362 and Ser374 in HK-2 cells treated with CdCl{sub 2}. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)-including extracellular signal-regulated protein kinase (ERK), c-Jun NH{sub 2}-terminal kinase, and p38-increased after CdCl{sub 2} exposure, whereas treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 suppressed the accumulation and phosphorylation of c-Fos. We mutated Ser362 to alanine (S362A), Ser374 to alanine (S374A), and both residues to alanines (S362A/S374A) to inhibit potential phosphorylation of c-Fos at these sites. S374A or double S362A/S374A mutations reduced c-Fos level markedly, but S362A mutation did not. On the other hand, S362A/S374A mutations induced a more pronounced reduction in c-Fos DNA-binding activity than S374A mutation. These results suggest that while Ser374 phosphorylation seems to play a role in c-Fos stabilization, phosphorylation at two C-terminal serine residues is required for the transcriptional activation of c-Fos in HK-2 cells treated with CdCl{sub 2}.

  17. Constituents of Cryptotaenia japonica Inhibit Melanogenesis via CREB- and MAPK-Associated Signaling Pathways in Murine B16 Melanoma Cells.

    PubMed

    Seong, Zuh-Kyung; Lee, Sung-Yoon; Poudel, Amrit; Oh, Sei-Ryang; Lee, Hyeong-Kyu

    2016-09-28

    Melanin plays an important role in protecting the skin against ultraviolet light and is responsible for skin color. However, overproduction of melanin is related to several skin disorders, such as age spots, freckles, café au lait spots, Becker's nevus and other hyperpigmentation syndromes. The aim of this study was to identify the effects of kaempferol-7-O-β-d-glucuronide (K7G) and tilianin, isolated from Cryptotaenia japonica, on melanogenesis and their mechanisms of action in murine B16 melanoma cells. The α-melanocyte-stimulating hormone (α-MSH)-induced melanin production was significantly inhibited by K7G and tilianin in a dose-dependent manner. The effects of these compounds on the signaling pathway of melanogenesis were examined. K7G and tilianin downregulated the expression of microphthalmia-associated transcription factor (MITF) and melanocyte-specific enzymes, i.e., tyrosinase and TRP1. These compounds also inhibited the phosphorylation of cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) in a dose-dependent manner. In addition, these compounds increased the phosphorylation of extracellular signal-regulated kinase (ERK) but decreased the phosphorylation of c-Jun N-terminal kinase (JNK) in B16 cells. Based on the above results, the anti-melanogenic effects of these compounds are caused by suppression of the MAPK signaling pathway through the down-regulation of α-MSH-induced CREB accumulation. This finding suggests that K7G and tilianin may be good candidates for further research to develop therapeutic agents for hyperpigmentation diseases.

  18. p38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis

    SciTech Connect

    Grethe, Simone; Coltella, Nadia; Di Renzo, Maria Flavia; Poern-Ares, M. Isabella . E-mail: isabella.ares@helsinki.fi

    2006-09-01

    Doxorubicin is the anthracycline with the widest spectrum of antitumor activity, and it has been shown that the antitumor activity is mediated in vivo by selective triggering of apoptosis in proliferating endothelial cells. We studied cultured human endothelial cells and observed that doxorubicin-induced apoptosis was mediated by p38 mitogen-activated protein kinase (MAPK). Doxorubicin-provoked apoptosis was significantly inhibited by expression of dominant negative p38 MAPK or pharmacological inhibition with SB203580. Furthermore, blocking phosphatidylinositol-3-kinase/Akt signaling significantly increased doxorubicin-induced caspase-3 activity and cell death, indicating that Akt is a survival factor in this system. Notably, we also found that doxorubicin-provoked apoptosis included p38 MAPK-mediated inhibition of Akt and Bad phosphorylation. Furthermore, doxorubicin-stimulated phosphorylation of Bad in cells expressing dominant negative p38 MAPK was impeded by the inhibition of PI3-K. In addition to the impact on Bad phosphorylation, doxorubicin-treatment caused p38 MAPK-dependent downregulation of Bcl-xL protein.

  19. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio)

    PubMed Central

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine’s metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol’s locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug. PMID:26366782

  20. Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation

    SciTech Connect

    Zhou, Yan; Ruan, Zheng; Zhou, Lili; Shu, Xugang; Sun, Xiaohong; Mi, Shumei; Yang, Yuhui; Yin, Yulong

    2016-01-22

    Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. - Highlights: • Dietary supplementation with chlorogenic acid (CGA) improved endotoxin-induced liver injury. • Chlorogenic acid enhances ATP increase and shifts energy metabolism, which is correlated with up-regulation AMPK and PGC-1α. • The possible mechanism of CGA on mitochondrial biogenesis was correlated with up-regulation AMPK and PGC-1α.

  1. Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance

    PubMed Central

    Petersen, Max C.; Madiraju, Anila K.; Gassaway, Brandon M.; Marcel, Michael; Nasiri, Ali R.; Butrico, Gina; Marcucci, Melissa J.; Zhang, Dongyan; Abulizi, Abudukadier; Zhang, Xian-Man; Philbrick, William; Hubbard, Stevan R.; Samuel, Varman T.; Rinehart, Jesse

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet–induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance. PMID:27760050

  2. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    PubMed

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  3. Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner.

    PubMed

    Watson, Ben R; White, Nathan A; Taylor, Kirk A; Howes, Joanna-Marie; Malcor, Jean-Daniel M; Bihan, Dominique; Sage, Stewart O; Farndale, Richard W; Pugh, Nicholas

    2016-01-01

    Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.

  4. Piper sarmentosum Roxb. produces antidepressant-like effects in rodents, associated with activation of the CREB-BDNF-ERK signaling pathway and reversal of HPA axis hyperactivity.

    PubMed

    Li, Qing; Qu, Fa-Lin; Gao, Yue; Jiang, Yi-Ping; Rahman, Khalid; Lee, Kuo-Hsiung; Han, Ting; Qin, Lu-Ping

    2017-03-06

    There are many plants of genus Piper which have been reported to induce antidepressant-like effects, Piper sarmentosum (PS) is one of them. PS is a Chinese herbal medicine and a traditional edible vegetable. In the present study, the antidepressant-like effects of PS extracts and the ethyl acetate fraction of PS extracts (PSY) were assessed using the open field test (OFT), forced swimming test (FST), and tail suspension test (TST) in mice. Furthermore, we applied a 4 consecutive weeks of chronic unpredictable mild stress (CUMS) as a model of depression in rats, followed by a sucrose preference test. Then we examined the possible mechanisms of this action. The activity of the hypothalamic-pituitary-adrenal (HPA) axis was evaluated by detecting the serum corticosterone (CORT) concentrations, and the protein expression levels of brain-derived neurotrophic factor (BDNF), the phosphorylated form CREB and ERK1/2 were detected by qRT-PCR or Western blot. The results showed that PS extracts (100, 200mg/kg) and PSY (12.5, 25, 50mg/kg) treatment produced antidepressant-like effects in mice similar to fluoxetine (20mg/kg), indicated by the reduced immobility time in the FST and TST, while both had no influence on the locomotor activity in the OFT. PSY treatment significantly increased sucrose preference and reduced serum CORT levels in CUMS rats. Moreover, PSY up-regulated BDNF protein levels, and increased CREB and ERK phosphorylation levels in the hippocampus on CUMS rats. These findings suggest that the antidepressant-like effects of PS extracts and PSY are mediated, at least in part, by modulating HPA axis, BDNF, CREB and ERK phosphorylation and expression in the hippocampus. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  5. p38α MAPK is required for arsenic-induced cell transformation.

    PubMed

    Kim, Hong-Gyum; Shi, Chengcheng; Bode, Ann M; Dong, Zigang

    2016-05-01

    Arsenic exposure has been reported to cause neoplastic transformation through the activation of PcG proteins. In the present study, we show that activation of p38α mitogen-activated protein kinase (MAPK) is required for arsenic-induced neoplastic transformation. Exposure of cells to 0.5 μM arsenic increased CRE and c-Fos promoter activities that were accompanied by increases in p38α MAPK and CREB phosphorylation and expression levels concurrently with AP-1 activation. Introduction of short hairpin (sh) RNA-p38α into BALB/c 3T3 cells markedly suppressed arsenic-induced colony formation compared with wildtype cells. CREB phosphorylation and AP-1 activation were decreased in p38α knockdown cells after arsenic treatment. Arsenic-induced AP-1 activation, measured as c-Fos and CRE promoter activities, and CREB phosphorylation were attenuated by p38 inhibition in BALB/c 3T3 cells. Thus, p38α MAPK activation is required for arsenic-induced neoplastic transformation mediated through CREB phosphorylation and AP-1 activation.

  6. MeCP2 phosphorylation limits psychostimulant-induced behavioral and neuronal plasticity.

    PubMed

    Deng, Jie V; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C; Greenberg, Michael E; Kenny, Paul J; Calakos, Nicole; West, Anne E

    2014-03-26

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors.

  7. MeCP2 Phosphorylation Limits Psychostimulant-Induced Behavioral and Neuronal Plasticity

    PubMed Central

    Deng, Jie V.; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C.; Greenberg, Michael E.; Kenny, Paul J.; Calakos, Nicole

    2014-01-01

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors. PMID:24671997

  8. Tyrosine hydroxylase phosphorylation after naloxone-induced morphine withdrawal in the left ventricle.

    PubMed

    Almela, Pilar; Victoria Milanés, Maria; Luisa Laorden, Maria

    2009-07-01

    Our previous studies have shown that morphine withdrawal induced hyperactivity of cardiac noradrenergic pathways. The purpose of the present study was to evaluate the effects of morphine withdrawal on site-specific tyrosine hydroxylase (TH) phosphorylation in the rat left ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg/kg, s.c.). TH phosphorylation was determined by quantitative blot immunolabelling using phosphorylation state-specific antibodies. Ninety min after naloxone administration to morphine-dependent rats there was an increase in phospho-Ser40-TH (139.0 +/- 13%, P < 0.05) and Ser31-TH (135.5 +/- 11%, P < 0.05) in the left ventricle which is associated with both an increase in total TH levels (114.4 +/- 4.6%, P < 0.05, P < 0.01) and an enhancement of TH activity (51.0 +/- 11 dm/microg protein, P < 0.001). When HA-1004 (40 nmol/day), inhibitor of cyclic AMP dependent protein kinase (PKA) was infused, concomitantly with morphine, it diminished the increase in noradrenaline (NA) turnover, total TH expression (95.76 +/- 4.1 %, P < 0.01) and TH phosphorylation at Ser40 (85.5 +/- 11%, P < 0.01) in morphine-withdrawn rats. In addition, we showed that the ability of morphine withdrawal to stimulate phosphorylation at serine 31 is reduced (101.7 +/- 7.7%, P < 0.05) by SL327 (100 mg/kg, i.p.), an inhibitor of extracellular signal-regulated kinase (ERK) activation. The present findings demonstrate that the enhancement of total TH expression and the increase of the phosphorylation state of TH during morphine withdrawal are dependent on PKA and ERK and suggest that these transduction pathways might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine- withdrawal.

  9. Effects of Aluminium on Long-Term Memory in Rats and on SIRT1 Mediating the Transcription of CREB-Dependent Gene in Hippocampus.

    PubMed

    Yan, Dongying; Jin, Cuihong; Cao, Yang; Wang, Lulu; Lu, Xiaobo; Yang, Jinghua; Wu, Shengwen; Cai, Yuan

    2017-10-01

    Epidemiological investigations have shown that aluminium (Al) is an important neurotoxicant which can be absorbed by organisms via various routes. Previous studies have confirmed that exposure to Al could cause neurodegenerative diseases, decline CREB phosphorylation and then down-regulate the transcription and protein expression of its target genes including BDNF. However, recent studies revealed that CREB activation alone was far from enough to activate the expression of long-term memory (LTM)-related genes; there might be other regulatory factors involved in this process. Several studies showed that TORC1 might be involved in regulating the transcription of downstream target genes as well. Also, TORC1 could be mediated by SIRT1 during the formation of LTM. However, the role of CREB regulating system in Al-induced LTM impairment was still not utterly elucidated till now. This study was designed to establish the rat model of subchronic Al exposure to observe the neuroethology, regulatory factor levels and molecular biological alterations in hippocampal cells. The results showed that, with the increasing AlCl3 dose, blood Al content increased gradually; morphology of the hippocampus and neuronal ultrastructure were aberrant; in the Morris water maze test, the escape latency and distance travelled became longer, swimming traces turned more complicated in the place navigation test; intracellular Ca(2+) , cAMP levels declined significantly in AlCl3 -treated rats, followed by abated nuclear translocation of TORC1 and decreased SIRT1, TORC1 and pCREB levels. These results indicate that SIRT1 and TORC1 might play an important mediating role in Al-induced LTM impairment. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  10. Mixture of Peanut Skin Extract and Fish Oil Improves Memory in Mice via Modulation of Anti-Oxidative Stress and Regulation of BDNF/ERK/CREB Signaling Pathways

    PubMed Central

    Xiang, Lan; Cao, Xue-Li; Xing, Tian-Yan; Mori, Daisuke; Tang, Rui-Qi; Li, Jing; Gao, Li-Juan; Qi, Jian-Hua

    2016-01-01

    Long-term use of fish oil (FO) is known to induce oxidative stress and increase the risk of Alzheimer’s disease in humans. In the present study, peanut skin extract (PSE), which has strong antioxidant capacity, was mixed with FO to reduce its side effects while maintaining its beneficial properties. Twelve-week Institute of Cancer Research (ICR) mice were used to conduct animal behavior tests in order to evaluate the memory-enhancing ability of the mixture of peanut skin extract and fish oil (MPF). MPF significantly increased alternations in the Y-maze and cognitive index in the novel object recognition test. MPF also improved performance in the water maze test. We further sought to understand the mechanisms underlying these effects. A significant decrease in superoxide dismutase (SOD) activity and an increase in malonyldialdehyde (MDA) in plasma were observed in the FO group. The MPF group showed reduced MDA level and increased SOD activity in the plasma, cortex and hippocampus. Furthermore, the gene expression levels of brain-derived neurotrophic factor (BDNF) and cAMP responsive element-binding protein (CREB) in the hippocampus were increased in the MPF group, while phosphorylation of protein kinase B (AKT), extracellular signal-regulated kinase (ERK) and CREB in the hippocampus were enhanced. MPF improves memory in mice via modulation of anti-oxidative stress and activation of BDNF/ERK/CREB signaling pathways. PMID:27136583

  11. Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells

    PubMed Central

    Wang, Jian-Fa; Fu, Shou-Peng; Li, Su-Nan; Hu, Zhong-Ming; Xue, Wen-Jing; Li, Zhi-Qiang; Huang, Bing-Xu; Lv, Qing-Kang; Liu, Ju-Xiong; Wang, Wei

    2013-01-01

    Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake. PMID:24177567

  12. Phosphatidylinositol 3-kinase and calcium-activated transcription pathways are required for VLDL-induced smooth muscle cell proliferation.

    PubMed

    Lipskaia, Larissa; Pourci, Marie-Luce; Deloménie, Claudine; Combettes, Laurent; Goudounèche, Dominique; Paul, Jean-Louis; Capiod, Thierry; Lompré, Anne-Marie

    2003-05-30

    Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca2+ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca2+ release was inhibited and the IP3-sensitive Ca2+ release induced by ATP (10 micromol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3beta in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca2+-induced Ca2+ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.

  13. Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation.

    PubMed

    Paddock, Cathy; Lytle, Betsy L; Peterson, Francis C; Holyst, Trudy; Newman, Peter J; Volkman, Brian F; Newman, Debra K

    2011-06-02

    Immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors inhibit cellular responsiveness to immunoreceptor tyrosine-based activation motif (ITAM)-linked receptors. Although tyrosine phosphorylation is central to the initiation of both inhibitory ITIM and stimulatory ITAM signaling, the events that regulate receptor phosphorylation are incompletely understood. Previous studies have shown that ITAM tyrosines engage in structure-inducing interactions with the plasma membrane that must be relieved for phosphorylation to occur. Whether ITIM phosphorylation is similarly regulated and the mechanisms responsible for release from plasma membrane interactions to enable phosphorylation, however, have not been defined. PECAM-1 is a dual ITIM-containing receptor that inhibits ITAM-dependent responses in hematopoietic cells. We found that the PECAM-1 cytoplasmic domain is unstructured in an aqueous environment but adopts an α-helical conformation within a localized region on interaction with lipid vesicles that mimic the plasma membrane. The lipid-interacting segment contains the C-terminal ITIM tyrosine and a serine residue that undergo activation-dependent phosphorylation. The N-terminal ITIM is excluded from the lipid-interacting segment, and its phosphorylation is secondary to phosphorylation of the membrane-interacting C-terminal ITIM. On the basis of these findings, we propose a novel model for regulation of inhibitory signaling by ITIM-containing receptors that relies on reversible plasma membrane interactions and sequential ITIM phosphorylation.

  14. Effects of acute cocaine on ERK and DARPP-32 phosphorylation pathways in the caudate-putamen of Fischer rats

    PubMed Central

    Sun, Wei-Lun; Zhou, Luyi; Hazim, Ruhal; Quinones-Jenab, Vanya; Jenab, Shirzad

    2010-01-01

    Activation of extracellular signal-regulated kinase (ERK) and dopamine- and cAMP-regulated phosphoprotein (DARPP-32) pathways has been implicated in biochemical and behavioral effects induced by various drugs of abuse. In this study, we investigated the phosphorylation pathways of these two proteins in response to acute cocaine administration. A single cocaine administration (30 mg/kg) increased ERK-mediated signaling proteins, phosphoryation of cAMP response element-binding protein (CREB) kinase, pp90 ribosomal S6 kinase (RSK), and c-Fos protein levels in the caudate/putamen of Fischer rats. Acute cocaine administration also induced phosphorylation of the striatal-enriched protein tyrosine phosphatase (STEP) and decreased the phosphorylation of DARPP-32 protein at the Thr-75 site. The phosphorylation states of these inhibitors of ERK and DARPP-32 proteins may thus contribute to the effects of cocaine on ERK- and DARPP-32-mediated cascades, on gene expression and on behaviors. PMID:17920048

  15. The declined phosphorylation of Hsp27 in rat cardiac muscle after simulated microgravity induced by hindlimb unloading

    NASA Astrophysics Data System (ADS)

    Yuan, Ming; Jiang, Shizhong; Li, Zhili; Yuan, Min; Dong, Weijun

    Many studies have shown that simulated microgravity induced by hindlimb unloading can decrease the contractility of rat cardiac muscle however the mechanisms responsible for which remain unclear Actin polymerization which can be regulated by Hsp27 has important role in the transmission of stress force during the contraction of cardiac muscle In this study western blot analysis was used to detect the expression of Hsp27 and phosphorylated Hsp27 FAK and phosphorylated FAK P38 MAPK and phosphorylated P38 MAPK in rat cardiac muscle after 14d hindlimb unloading The results showed that the phosphorylation levels of both Hsp27 and P38 MAPK were declined significantly which may decrease actin polymerization and inhibit the transmission of stress force during the contraction of rat cardiac muscle after hindlimb unloading However the phosphorylation level of FAK was not declined significantly in cardiac muscle The results suggested that the declined phosphorylation level of Hsp27 which may be ascribable to the decline of contractility of rat cardiac muscle after 14d hindlimb unloading may be induced by the declined phosphorylation level of P38 MAPK but not phosphorylation level of FAK

  16. Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism.

    PubMed

    Wu, Hongling; Li, Ting; Zeng, Musheng; Peng, Tao

    2012-04-01

    The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.

  17. The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes

    PubMed Central

    Sugden, Chris; Urbaniak, Michael D.; Araki, Tsuyoshi; Williams, Jeffrey G.

    2015-01-01

    Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1–controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca2+/calmodulin–dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555. PMID:25518940

  18. Phosphorylation of p53 by LRRK2 induces microglial tumor necrosis factor α-mediated neurotoxicity.

    PubMed

    Ho, Dong Hwan; Seol, Wongi; Eun, Jin Hwan; Son, Il-Hong

    2017-01-22

    Leucine-rich repeat kinase (LRRK2), a major causal gene of Parkinson's disease (PD), functions as a kinase. The most prevalent mutation of LRRK2 is G2019S. It exhibits increased kinase activity compared to the wildtype LRRK2. Previous studies have shown that LRRK2 can phosphorylate p53 at T304 and T377 of threonine-X-arginine (TXR) motif in neurons. Reduction of LRRK2 expression or inhibition of LRRK2 kinase activity has been shown to be able to alleviate LPS-induced neuroinflammation in microglia cells. In this study, we found that LRRK2 could also phosphorylate p53 in microglia model BV2 cells. Transfection of BV2 with phosphomimetic p53 T304/377D significantly increased the secretion of pro-inflammatory cytokine TNFα compared to BV2 transfected with p53 wild type after LPS treatment. In addition, conditioned media from these transfected cells increased the death of dopaminergic neuronal SN4741 cells. Moreover, such neurotoxic effect was rescued by co-treatment with the conditioned media and etanercept, a TNFα blocking antibody. Furthermore, TNFα secretion was significantly increased in primary microglia derived from G2019S transgenic mice treated with LPS compared to that in cells derived from their littermates. These results suggest that LRRK2 kinase activity in microglia can contribute to neuroinflammation in PD via phosphorylating p53 at T304 and T377 site.

  19. Depletion of the Adaptor Protein NCK Increases UV-Induced p53 Phosphorylation and Promotes Apoptosis

    PubMed Central

    Errington, Timothy M.; Macara, Ian G.

    2013-01-01

    The cellular response to DNA damage requires the coordination of many proteins involved in diverse molecular processes. Discrete molecular pathways are becoming increasingly well understood, but the interconnectivity and coordination of multiple pathways remains less clear. We now show that NCK, an adapter protein involved in cytoskeletal responses to tyrosine kinase receptor signaling, accumulates in the nucleus in response to DNA damage and this translocation can be blocked by specific inhibition of the ATR protein kinase. Strikingly, HeLa cells depleted of NCK undergo apoptosis shortly after UV irradiation, as monitored by caspase-3 cleavage and PARP cleavage. This rapid, hyperactive apoptosis in NCK depleted cells might be p53 dependent, because loss of NCK also increased UV-induced p53 phosphorylation. Importantly, depletion of SOCS7, which is necessary for NCK nuclear translocation, phenocopies NCK depletion, indicating the nuclear accumulation of NCK is responsible for these molecular events. There are two NCK isoforms that have mostly redundant functions, and although NCK2 appears to have a greater contribution, depletion of NCK1 or NCK2, led to increased p53 phosphorylation and early apoptosis after UV exposure. These data reveal a novel function for NCK in regulating p53 phosphorylation and apoptosis, and provide evidence for interconnectedness of growth factor signaling proteins and the DNA damage response. PMID:24086708

  20. Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

    PubMed

    Werner, David F; Porcu, Patrizia; Boyd, Kevin N; O'Buckley, Todd K; Carter, Jenna M; Kumar, Sandeep; Morrow, A Leslie

    2016-04-01

    GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

  1. CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

    PubMed Central

    Xu, Shangcheng; Wang, Pei; Zhang, Huiliang; Gong, Guohua; Gutierrez Cortes, Nicolas; Zhu, Weizhong; Yoon, Yisang; Tian, Rong; Wang, Wang

    2016-01-01

    Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca2+/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart. PMID:27739424

  2. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  3. The novel mechanism of rotenone-induced α-synuclein phosphorylation via reduced protein phosphatase 2A activity.

    PubMed

    Wang, Yi; Liu, Jia; Chen, Min; Du, Tingting; Duan, Chunli; Gao, Ge; Yang, Hui

    2016-06-01

    Rotenone has been shown to induce many parkinsonian features and has been widely used in chemical models of Parkinson's disease (PD). Its use is closely associated with α-synuclein (α-syn) phosphorylation both in vivo and in vitro. However, the mechanisms whereby rotenone regulates α-syn phosphorylation remain unknown. Protein phosphatase 2A (PP2A) has been shown to play an important role in α-syn dephosphorylation. We therefore investigated if rotenone caused α-syn phosphorylation by down-regulation of PP2A activity in mice. Rotenone increased the phosphorylation of α-syn at Ser129, consistent with the inhibition of PP2A activity by increased phosphorylation of tyrosine 307 at the catalytic subunit of PP2A (pTyr307 PP2Ac). We further explored the interactions among rotenone, PP2A, and α-syn in SK-N-SH cells and primary rat cortical neurons. Rotenone inhibited PP2A activity via phosphorylation of PP2Ac at Tyr307. The reduction in PP2A activity and rotenone cytotoxicity were reversed by treatment with the PP2A agonist, C2 ceramide, and the Src kinase inhibitor, SKI606. Immunoprecipitation experiments showed that rotenone induced an increase in calmodulin-Src complex in SK-N-SH cells, thus activating Src kinase, which in turn phosphorylated PP2A at Tyr307 and inhibited its activity. C2 ceramide and SKI606 significantly reversed the rotenone-induced phosphorylation and aggregation of α-syn by increasing PP2A activity. These results demonstrate that rotenone-reduced PP2A activity via Src kinase is involved in the phosphorylation of α-syn. These findings clarify the novel mechanisms whereby rotenone can induce PD. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Persistent phosphorylation at specific H3 serine residues involved in chemical carcinogen-induced cell transformation.

    PubMed

    Zhu, Xiaonian; Li, Daochuan; Zhang, Zhengbao; Zhu, Wei; Li, Wenxue; Zhao, Jian; Xing, Xiumei; He, Zhini; Wang, Shan; Wang, Fangping; Ma, Lu; Bai, Qing; Zeng, Xiaowen; Li, Jie; Gao, Chen; Xiao, Yongmei; Wang, Qing; Chen, Liping; Chen, Wen

    2017-05-01

    Identification of aberrant histone H3 phosphorylation during chemical carcinogenesis will lead to a better understanding of the substantial roles of histone modifications in cancer development. To explore whether aberrant H3 phosphorylation contributes to chemical carcinogenesis, we examined the dynamic changes of H3 phosphorylation at various residues in chemical carcinogen-induced transformed human cells and human cancers. We found that histone H3 phosphorylation at Ser10 (p-H3S10) and Ser28 (p-H3S28) was upregulated by 1.5-4.8 folds and 2.1-4.3 folds, respectively in aflatoxin B1 -transformed hepatocytes L02 cells (L02RT-AFB1 ), benzo(a)pyrene-transformed HBE cells (HBERT-BaP), and coke oven emissions-transformed HBE cells (HBERT-COE). The ectopic expression of histone H3 mutant (H3S10A or H3S28A) in L02 cells led to the suppression of an anchorage-independent cell growth as well as tumor formation in immunodeficient mice. In addition, an enhanced p-H3S10 was found in 70.6% (24/34) of hepatocellular carcinoma (HCC), and 70.0% (21/30) of primary lung cancer, respectively. Notably, we found that expression of H3 carrying a mutant H3S10A or H3S28A conferred to cells the ability to maintain a denser chromatin and resistance to induction of DNA damage and carcinogen-induced cell transformation. Particularly, we showed that introduction of a mutant H3S10A abolished the bindings of p-H3S10 to the promoter of DNA repair genes, PARP1 and MLH1 upon AFB1 treatment. Furthermore, we revealed that PP2A was responsible for dephosphorylation of p-H3S10. Taken together, these results reveal a key role of persistent H3S10 or H3S28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response (DDR) genes. © 2017 Wiley Periodicals, Inc.

  5. Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

    PubMed

    Hough, Chris; Radu, Maria; Doré, Jules J E

    2012-01-01

    The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

  6. Inflammatory cytokines induce phosphorylation and ubiquitination of prostate suppressor protein NKX3.1.

    PubMed

    Markowski, Mark C; Bowen, Cai; Gelmann, Edward P

    2008-09-01

    Inflammation of the prostate is a risk factor for the development of prostate cancer. In the aging prostate, regions of inflammatory atrophy are foci for prostate epithelial cell transformation. Expression of the suppressor protein NKX3.1 is reduced in regions of inflammatory atrophy and in preinvasive prostate cancer. Inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1beta accelerate NKX3.1 protein loss by inducing rapid ubiquitination and proteasomal degradation. The effect of TNF-alpha is mediated via the COOH-terminal domain of NKX3.1 where phosphorylation of serine 196 is critical for cytokine-induced degradation. Mutation of serine 196 to alanine abrogates phosphorylation at that site and the effect of TNF-alpha on NKX3.1 ubiquitination and protein loss. This is in contrast to control of steady-state NKX3.1 turnover, which is mediated by serine 185. Mutation of serine 185 to alanine increases NKX3.1 protein stability by inhibiting ubiquitination and doubling the protein half-life. A third COOH-terminal serine at position 195 has a modulating effect on both steady-state protein turnover and on ubiquitination induced by TNF-alpha. Thus, cellular levels of the NKX3.1 tumor suppressor are affected by inflammatory cytokines that target COOH-terminal serine residues to activate ubiquitination and protein degradation. Our data suggest that strategies to inhibit inflammation or to inhibit effector kinases may be useful approaches to prostate cancer prevention.

  7. Mineral induced phosphorylation of glycolate ion--a metaphor in chemical evolution

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Zhang, S.; Xu, Y.; Arrhenius, G.

    1997-01-01

    Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from l0(-3) m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from l0(-2) m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.

  8. Mineral induced phosphorylation of glycolate ion--a metaphor in chemical evolution

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Zhang, S.; Xu, Y.; Arrhenius, G.

    1997-01-01

    Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from l0(-3) m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from l0(-2) m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.

  9. Mineral induced phosphorylation of glycolate ion--a metaphor in chemical evolution.

    PubMed

    Kolb, V; Zhang, S; Xu, Y; Arrhenius, G

    1997-12-01

    Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from l0(-3) m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from l0(-2) m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.

  10. Pesticides induce spatial memory deficits with synaptic impairments and an imbalanced tau phosphorylation in rats.

    PubMed

    Chen, Ning-Ning; Luo, Dan-Ju; Yao, Xiu-Qing; Yu, Cong; Wang, Yi; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2012-01-01

    Pesticides are widely used in agriculture, and epidemiological studies suggest that pesticide exposure is a risk factor for Alzheimer's disease (AD), but the mechanisms are elusive. Here, we studied the effects of pesticide exposure on the cognitive ability and the underlying mechanisms in rats. Deltamethrin and carbofuran were administered respectively into the rats once a day for 28 days by gavage. We found that pesticide exposure induced spatial learning and memory deficits with a simultaneous decrease of N-methyl-D-aspartate receptor 1, synaptophysin, and synapsin I, all of which are memory-related synaptic proteins. Pesticide exposure also induced tau hyperphosphorylation at multiple AD-related phosphorylation sites with activation of glycogen synthase kinase-3β and inhibition of protein phosphatase-2A. Additionally, neuron loss in the hippocampus and cortex was observed upon administration of the pesticides. These results indicate that the pesticides exposure could induce AD-like pathology and cognitive abnormality in rats.

  11. Requirement for tyrosine phosphorylation in lipopolysaccharide-induced murine B-cell proliferation.

    PubMed Central

    Dearden-Badet, M T; Revillard, J P

    1993-01-01

    Bacterial lipopolysaccharide (LPS) induces a strong B-cell proliferative response with subsequent differentiation, through a complex signal transduction pathway. This process is known to be mediated through protein kinase C (PKC) translocation without Ca2+ mobilization. Here, we show that B-cell proliferative responses induced by five different LPS preparations, as well as by F(ab')2 anti-IgM antibodies, are inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A. In contrast, B-cell proliferation induced by the combination of phorbol 12-myristate 13-acetate (PMA) plus ionomycin was not influenced by treatment with either herbimycin A or genistein. These data indicate that tyrosine phosphorylation is required to initiate B-cell proliferation by LPS. PMID:8307617

  12. CREB Regulates Experience-Dependent Spine Formation and Enlargement in Mouse Barrel Cortex

    PubMed Central

    Pignataro, Annabella; Borreca, Antonella; Ammassari-Teule, Martine; Middei, Silvia

    2015-01-01

    Experience modifies synaptic connectivity through processes that involve dendritic spine rearrangements in neuronal circuits. Although cAMP response element binding protein (CREB) has a key function in spines changes, its role in activity-dependent rearrangements in brain regions of rodents interacting with the surrounding environment has received little attention so far. Here we studied the effects of vibrissae trimming, a widely used model of sensory deprivation-induced cortical plasticity, on processes associated with dendritic spine rearrangements in the barrel cortex of a transgenic mouse model of CREB downregulation (mCREB mice). We found that sensory deprivation through prolonged whisker trimming leads to an increased number of thin spines in the layer V of related barrel cortex (Contra) in wild type but not mCREB mice. In the barrel field controlling spared whiskers (Ipsi), the same trimming protocol results in a CREB-dependent enlargement of dendritic spines. Last, we demonstrated that CREB regulates structural rearrangements of synapses that associate with dynamic changes of dendritic spines. Our findings suggest that CREB plays a key role in dendritic spine dynamics and synaptic circuits rearrangements that account for new brain connectivity in response to changes in the environment. PMID:26075101

  13. Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

    PubMed

    Suo, Satoshi; Ishiura, Shoichi

    2013-01-01

    Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB) is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

  14. CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor.

    PubMed

    Eraso-Pichot, A; Larramona-Arcas, R; Vicario-Orri, E; Villalonga, R; Pardo, L; Galea, E; Masgrau, R

    2017-03-01

    Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

  15. Interactions between CBP, NF-kappaB, and CREB in the lungs after hemorrhage and endotoxemia.

    PubMed

    Shenkar, R; Yum, H K; Arcaroli, J; Kupfner, J; Abraham, E

    2001-08-01

    The transcriptional regulatory factor nuclear factor (NF)-kappaB has a central role in modulating expression of proinflammatory mediators that are important in acute lung injury. In vitro studies have shown that competition between NF-kappaB and cAMP response element binding protein (CREB) for binding to the coactivator CREB-binding protein (CBP) is important in regulating transcriptional activity of these factors. In the present study, we examined in vivo interactions between CBP, CREB, and NF-kappaB in hemorrhage- or endotoxemia-induced acute lung injury. Association of CBP with CREB or the p65 subunit of NF-kappaB increased in the lungs after hemorrhage or endotoxemia. Inhibition of xanthine oxidase before hemorrhage, but not before endotoxemia, decreased p65-CBP interactions while increasing those between CREB and CBP. These alterations in CREB-CBP and p65-CBP interactions were functionally significant because xanthine oxidase inhibition before hemorrhage resulted in increased expression of the CREB-dependent gene c-Fos and decreased expression of macrophage inflammatory protein-2, a NF-kappaB-dependent gene. The present results show that the coactivator CBP has an important role in modulating transcription in vivo under clinically relevant pathophysiological conditions.

  16. PGE2 upregulates renin through E-prostanoid receptor 1 via PKC/cAMP/CREB pathway in M-1 Cells.

    PubMed

    Gonzalez, Alexis A; Salinas-Parra, Nicolas; Leach, Dan; Navar, L Gabriel; Prieto, Minolfa C

    2017-07-12

    During the early phase of angiotensin (ANG) II-dependent hypertension tubular prostaglandin E2 (PGE2) is increased. Renin synthesis and secretion in the collecting duct (CD) is upregulated by ANGII contributing to further intratubular ANGII formation. However, what happens first and whether the triggering mechanism is independent of tubular ANGII, remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular (JG) cells via E-prostanoid (EP) receptors through cAMP/CREB pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. M-1 CD cell line expressed EP1, EP3 and EP4 but not EP2. Dose response experiments in the presence of AT1 receptor blockade with candesartan demonstrated that 10-6 M PGE2 maximally increases renin mRNA (~4 fold) and prorenin/renin protein levels (~2 fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161,982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10 fold increase). To further evaluate the signaling pathway involved we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative (DN). Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation and augmentation of renin. Knockdown of EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through EP1 receptor via PKC/cAMP/CREB pathway. Therefore, we conclude that during early stages of ANGII-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANGII formation and further stimulation of renin. Copyright © 2017, American Journal of Physiology-Renal Physiology.

  17. Involvement of tyrosine phosphorylation in HMG-CoA reductase inhibitor-induced cell death in L6 myoblasts.

    PubMed

    Mutoh, T; Kumano, T; Nakagawa, H; Kuriyama, M

    1999-02-05

    Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, causes myopathy in rabbits and kills L6 myoblasts. The present study was designed to elucidate the molecular mechanism of HCRI-induced cell death. We have demonstrated that simvastatin induces the tyrosine phosphorylation of several cellular proteins within 10 min. These phosphorylations were followed by apoptosis, as evidenced by the occurrence of internucleosomal DNA fragmentation and by morphological changes detected with Nomarski optics. Simvastatin-induced cell death was prevented by tyrosine kinase inhibitors. The MTT assay revealed that the addition of mevalonic acid into the culture medium partially inhibited simvastatin-induced cell death. Thus, these results suggested that protein tyrosine phosphorylation might play an important role in the intracellular signal transduction pathway mediating the HCRI-induced death of myoblasts.

  18. Wnt3A Induces GSK-3β Phosphorylation and β-Catenin Accumulation Through RhoA/ROCK.

    PubMed

    Kim, Jae-Gyu; Kim, Myoung-Ju; Choi, Won-Ji; Moon, Mi-Young; Kim, Hee-Jun; Lee, Jae-Yong; Kim, Jaebong; Kim, Sung-Chan; Kang, Seung Goo; Seo, Goo-Young; Kim, Pyeung-Hyeun; Park, Jae-Bong

    2017-05-01

    In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

  19. Interleukin-6 autocrine signaling mediates melatonin MT(1/2) receptor-induced STAT3 Tyr(705) phosphorylation.

    PubMed

    Lau, Winnie W I; Ng, Johnson K Y; Lee, Maggie M K; Chan, Anthony S L; Wong, Yung H

    2012-05-01

    Melatonin receptors have previously been shown to elicit cellular signaling through the hematopoietic-specific G protein, G(16) . In the present study, we show that this functional coupling elicited biphasic stimulatory phosphorylation on STAT3 in recombinant MT(1) /Gα(16) cells and native Jurkat T cells (endogenously expressing MT(1) and Gα(16) ), with maximal Ser(727) phosphorylation occurring at 15min, while marked Tyr(705) phosphorylation became detectable only upon agonist treatment for 4 hr or more. By employing signal transducer and activator of transcription 3 (STAT3) phosphorylation-resistant mutants (STAT3-Y705F and STAT3-S727A), we further showed that the receptor-mediated STAT3 phosphorylations at Ser(727) and Tyr(705) were independent of each other. Results obtained from fractionation of 2-IMT-induced cells revealed that the Ser(727) and Tyr(705) phosphorylations were spatially distinct, with the former mainly situated in mitochondria and cytosol, while the latter was predominantly located in the nucleus. Further experiments revealed that the agonist-induced STAT3 phosphorylation at Tyr(705) was significantly suppressed by pretreatment with cycloheximide (a ribosome inhibitor), suggesting that de novo protein synthesis might play a critical role for this response. Using conditioned media obtained from 2-IMT-treated MT(1) /Gα(16) cells, multiplex immunoassays revealed that prolonged agonist treatment led to elevated productions of IL-6, GM-CSF and CXCL-8. Antibody against IL-6, but not those for GM-CSF and CXCL-8, effectively abolished the agonist-induced STAT3 Tyr(705) phosphorylation, suggesting the involvement of IL-6 in melatonin receptor-mediated STAT3 activation. Our results demonstrate that melatonin receptor/Gα(16) coupling is capable of triggering the production of cytokines including IL-6, and this autocrine loop may account for the subsequent STAT3 phosphorylation at Tyr(705) . © 2011 John Wiley & Sons A/S.

  20. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    SciTech Connect

    Pan, Xiao; Whitten, Douglas A.; Wu, Ming; Chan, Christina; Wilkerson, Curtis G.; Pestka, James J.

    2013-04-15

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

  1. Over-Expression of CREB in the Nucleus Accumbens Shell Increases Cocaine Reinforcement in Self-Administering Rats

    PubMed Central

    Larson, Erin B.; Graham, Danielle L.; Arzaga, Rose R.; Buzin, Nicole; Webb, Joseph; Green, Thomas A.; Bass, Caroline E.; Neve, Rachael L.; Terwilliger, Ernest F.; Nestler, Eric J.; Self, David W.

    2011-01-01

    Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we utilized viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed-ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to post-acquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Down-regulating endogenous CREB in NAc shell by expressing an shRNA reduced cocaine reinforcement in similar tests, while over expression of a dominant negative CREBS133A mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes. PMID:22072694

  2. Prostanoid EP1 receptors mediate up-regulation of the orphan nuclear receptor Nurr1 by cAMP-independent activation of protein kinase A, CREB and NF-κB

    PubMed Central

    Ji, R; Sanchez, CM; Chou, CL; Chen, XB; Woodward, DF; Regan, JW

    2012-01-01

    BACKGROUND AND PURPOSE Prostaglandin E2 (PGE2) stimulation of the G protein-coupled prostanoid EP1 receptor was found to up-regulate the expression of Nur-related factor 1 (Nurr1) (NR4A2), a transcription factor in the NR4A subfamily of nuclear receptors. The present studies characterize the molecular mechanism of this up-regulation. EXPERIMENTAL APPROACH The expression of Nurr1 was examined by immunoblot analysis, the polymerase chain reaction and reporter gene assays in human embryonic kidney (HEK) cells stably expressing the recombinant EP1 receptor and in SH-SY5Y neuroblastoma cells expressing endogenous EP1 receptors. Signalling pathway inhibitors were used to examine the roles of Rho, PKA, the cAMP response element binding protein (CREB) and NF-κB on the PGE2 stimulated up-regulation of Nurr1. CREB and NF-κB signalling were also examined by immunoblot analysis and reporter gene assays. KEY RESULTS The EP1 receptor mediated up-regulation of Nurr1 was blocked with inhibitors of Rho, PKA, NF-κB and CREB; but PGE2 failed to significantly stimulate intracellular cAMP formation. PGE2 stimulation of the EP1 receptor induced the phosphorylation and activation of CREB and NF-κB, which could be blocked by inhibition of PKA. CONCLUSIONS AND IMPLICATIONS PGE2 stimulation of the human EP1 receptor up-regulates the expression of Nurr1 by a mechanism involving the sequential activation of the Rho, PKA, CREB and NF-κB signalling pathways. EP1 receptors are implicated in tumorigenesis and the up-regulation of Nurr1 may underlie the anti-apoptotic effects of PGE2. PMID:22188298

  3. Prostanoid EP₁ receptors mediate up-regulation of the orphan nuclear receptor Nurr1 by cAMP-independent activation of protein kinase A, CREB and NF-κB.

    PubMed

    Ji, R; Sanchez, C M; Chou, C L; Chen, X B; Woodward, D F; Regan, J W

    2012-06-01

    Prostaglandin E(2) (PGE(2)) stimulation of the G protein-coupled prostanoid EP(1) receptor was found to up-regulate the expression of Nur-related factor 1 (Nurr1) (NR4A2), a transcription factor in the NR4A subfamily of nuclear receptors. The present studies characterize the molecular mechanism of this up-regulation. The expression of Nurr1 was examined by immunoblot analysis, the polymerase chain reaction and reporter gene assays in human embryonic kidney (HEK) cells stably expressing the recombinant EP(1) receptor and in SH-SY5Y neuroblastoma cells expressing endogenous EP(1) receptors. Signalling pathway inhibitors were used to examine the roles of Rho, PKA, the cAMP response element binding protein (CREB) and NF-κB on the PGE(2) stimulated up-regulation of Nurr1. CREB and NF-κB signalling were also examined by immunoblot analysis and reporter gene assays. The EP(1) receptor mediated up-regulation of Nurr1 was blocked with inhibitors of Rho, PKA, NF-κB and CREB; but PGE(2) failed to significantly stimulate intracellular cAMP formation. PGE(2) stimulation of the EP1 receptor induced the phosphorylation and activation of CREB and NF-κB, which could be blocked by inhibition of PKA. PGE(2) stimulation of the human EP(1) receptor up-regulates the expression of Nurr1 by a mechanism involving the sequential activation of the Rho, PKA, CREB and NF-κB signalling pathways. EP(1) receptors are implicated in tumorigenesis and the up-regulation of Nurr1 may underlie the anti-apoptotic effects of PGE(2) . © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  4. COMPARATIVE ANALYSES OF DIFFERENTIALLY-INDUCED TCR-MEDIATED PHOSPHORYLATION PATHWAYS IN T LYMPHOMA CELLS

    PubMed Central

    Ortiz, Serina; Lee, Wenhui; Smith, David; Forman, Stephen J.; Lee, Terry D.; Liu, Chih-Pin

    2011-01-01

    Activation of T lymphoma cells expressing Syk, but not ZAP-70 tyrosine kinase, has been shown to negatively regulate cell activation and activation induced cell death (AICD), perhaps due to differential induction of tyrosine phosphorylation modified proteins. To better understand the role of these proteins and their associated molecules/pathways, we studied a previously described model of T lymphoma cells expressing either a kinase-activated chimeric Syk or ZAP-70 genetically linked to TCR ζ chain (Z/Syk or Z/ZAP cells, respectively). To help identify molecules and pathways linked to cell activation or AICD, a comparative semi-quantitative proteomics-based approach was utilized to analyze tyrosine phosphorylated protein immunoprecipitates from 2 min short-term activated Z/Syk or Z/ZAP cells. Using the resulting bioinformatics datasets, we identified several differentially immunoprecipitated proteins that could be validated biochemically. More tyrosine-phosphorylated and phosphotyrosine-associated proteins were found in Z/Syk than in Z/ZAP cells. Proteins involved in different unique functional pathways were induced in these cells and showed altered intermolecular interactions in varied pathways. Remarkably, 41% of differentially identified proteins in Z/Syk cells belonged to cell cycle or vesicle/trafficking pathways. In contrast, 21% of such proteins in Z/ZAP cells belonged to metabolism pathways. Therefore, molecular pathways involved in post-translational modifications linked to distinct cellular/physiological functions are differentially activated, which may contribute to varied activation and AICD responses of these cells. In summary, we identified proteins belonging to novel differentially activated pathways involved in TCR-mediated signaling, which may be targets for regulating activation and AICD of T lymphoma cells and for potential cancer therapy. PMID:21127342

  5. Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa.

    PubMed

    Leemans, Bart; Gadella, Bart M; Sostaric, Edita; Nelis, Hilde; Stout, Tom A E; Hoogewijs, Maarten; Van Soom, Ann

    2014-07-01

    Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during which it transpired that the highest density (per mm(2)) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm. © 2014 by the Society for the Study of Reproduction, Inc.

  6. ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1

    PubMed Central

    Galbraith, Matthew D.; Saxton, Janice; Li, Li; Shelton, Samuel J.; Zhang, Hongmei; Espinosa, Joaquin M.; Shaw, Peter E.

    2013-01-01

    The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression. PMID:24049075

  7. ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1.

    PubMed

    Galbraith, Matthew D; Saxton, Janice; Li, Li; Shelton, Samuel J; Zhang, Hongmei; Espinosa, Joaquin M; Shaw, Peter E

    2013-12-01

    The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown o