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Sample records for erk1 mediates acquired

  1. ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration.

    PubMed

    Jaldety, Yael; Breitbart, Haim

    2015-10-01

    Mammalian sperm acquire fertilization capacity after residing in the female reproductive tract for a few hours in a process called capacitation. Only capacitated sperm can bind the zona pellucida (ZP) of the egg and undergo the acrosome reaction, a process that allows penetration and fertilization. Extracellular signal regulated kinase (ERK1/2) mediates signalling in many cell types, however its role in sperm function is largely unknown. Here we show that ERK1/2 is highly phosphorylated/activated after a short incubation of mouse sperm under capacitation conditions and that this phosphorylation is reduced after longer incubation. Further phosphorylation was observed upon addition of crude extract of egg ZP or epidermal growth factor (EGF). The mitogen-activated ERK-kinase (MEK) inhibitor U0126 abolished ERK1/2 phosphorylation, in vitro fertilization rate and the acrosome reaction induced by ZP or EGF but not by the Ca2+-ionophore A23187. Moreover, inhibition of ERK1/2 along the capacitation process diminished almost completely the sperm's ability to go through the acrosome reaction, while inhibition at the end of capacitation attenuated the acrosome reaction rate by only 45%. The fact that the acrosome reaction, induced by the Ca2+ -ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca2+] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca2+ elevation and the subsequent occurrence of the acrosome reaction.

  2. Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

    PubMed

    Feng, Xiuyan; Zhang, Yiqian; Shao, Ningjun; Wang, Yanhui; Zhuang, Zhizhi; Wu, Ping; Lee, Matthew J; Liu, Yingli; Wang, Xiaonan; Zhuang, Jieqiu; Delpire, Eric; Gu, Dingying; Cai, Hui

    2015-05-15

    Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

  3. Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2*

    PubMed Central

    Wiemhoefer, Anne; Stargardt, Anita; van der Linden, Wouter A.; Renner, Maria C.; van Kesteren, Ronald E.; Stap, Jan; Raspe, Marcel A.; Tomkinson, Birgitta; Kessels, Helmut W.; Ovaa, Huib; Overkleeft, Herman S.; Florea, Bogdan; Reits, Eric A.

    2015-01-01

    Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function. PMID:26041847

  4. Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation.

    PubMed

    Medina, Manel G; Ledesma, Maria Dolores; Domínguez, Jorge E; Medina, Miguel; Zafra, Delia; Alameda, Francesc; Dotti, Carlos G; Navarro, Pilar

    2005-05-01

    Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA-/- mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology.

  5. Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells.

    PubMed

    Nowak, Grazyna; Clifton, Ginger L; Godwin, Malinda L; Bakajsova, Diana

    2006-10-01

    Previously, we showed that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-epsilon. This study examined the role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC-epsilon mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tert-butylhydroperoxide (TBHP) resulted in three- to fivefold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, alpha-ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, DeltaPsi(m), ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in noninjured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation, demonstrating that the protective effect of MEK1/2 inhibitor was not due to activation of Akt or inhibition of p38 pathway. Inhibition of PKC-epsilon did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC-epsilon mediate oxidant-induced mitochondrial dysfunction through independent pathways.

  6. Autoantibody-mediated cytotoxicity in paediatric opsoclonus-myoclonus syndrome is dependent on ERK-1/2 phophorylation.

    PubMed

    Fühlhuber, Verena; Bick, Sandra; Tschernatsch, Marlene; Dharmalingam, Backialakshmi; Kaps, Manfred; Preissner, Klaus T; Blaes, Franz

    2015-12-15

    Paediatric opsoclonus-myoclonus syndrome (OMS) is in 50% of the cases associated with a neuroblastoma as a paraneoplastic syndrome and is associated with surface-binding antibodies against cerebellar granular neurons (CGN). To evaluate possible pathogenic effects of these autoantibodies on CGN we examined their influence on the MAPKinase enzymes ERK-1/2 and p38 using flow cytometry and phospho-specific antibodies. OMS IgG but not IgG from neuroblastoma without OMS or healthy controls induced phosphorylation of ERK-1/2 in cerebellar granular neurons (p<0.01). No effect on p38 phosphorylation or on HEK293 control cell line could be detected. IgG-mediated phosphorylation of ERK-1/2 was associated with an increased cytotoxicity of CGN, which could be blocked by ERK-1/2 pathway inhibitor U0126. We here show that IgG-mediated anti-neuronal cytotoxicity in OMS is mediated by ERK-1/2 phosphorylation in CGN.

  7. Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

    PubMed

    Martínez-Mármol, Ramón; Comes, Núria; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vicente, Rubén; Pujadas, Lluís; Soriano, Eduardo; Sorkin, Alexander; Felipe, Antonio

    2016-04-01

    The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

  8. Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor.

    PubMed

    Lecat, Sandra; Belemnaba, Lazare; Galzi, Jean-Luc; Bucher, Bernard

    2015-07-01

    Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.

  9. A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis.

    PubMed

    Poderoso, Cecilia; Converso, Daniela P; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Galli, Soledad; Cornejo Maciel, Fabiana; Paz, Cristina; Carreras, María C; Poderoso, Juan J; Podestá, Ernesto J

    2008-01-16

    ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.

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

    PubMed Central

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

    2014-01-01

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

  11. Diosgenin promotes oligodendrocyte progenitor cell differentiation through estrogen receptor-mediated ERK1/2 activation to accelerate remyelination.

    PubMed

    Xiao, Lin; Guo, Dazhi; Hu, Chun; Shen, Weiran; Shan, Lei; Li, Cui; Liu, Xiuyun; Yang, Wenjing; Zhang, Weidong; He, Cheng

    2012-07-01

    Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is a prerequisite for remyelination after demyelination, and impairment of this process is suggested to be a major reason for remyelination failure. Diosgenin, a plant-derived steroid, has been implicated for therapeutic use in many diseases, but little is known about its effect on the central nervous system. In this study, using a purified rat OPC culture model, we show that diosgenin significantly and specifically promotes OPC differentiation without affecting the viability, proliferation, or migration of OPC. Interestingly, the effect of diosgenin can be blocked by estrogen receptor (ER) antagonist ICI 182780 but not by glucocorticoid and progesterone receptor antagonist RU38486, nor by mineralocorticoid receptor antagonist spirolactone. Moreover, it is revealed that both ER-alpha and ER-beta are expressed in OPC, and diosgenin can activate the extracellular signal-regulated kinase 1/2 (ERK1/2) in OPC via ER. The pro-differentiation effect of diosgenin can also be obstructed by the ERK inhibitor PD98059. Furthermore, in the cuprizone-induced demyelination model, it is demonstrated that diosgenin administration significantly accelerates/enhances remyelination as detected by Luxol fast blue stain, MBP immunohistochemistry and real time RT-PCR. Diosgenin also increases the number of mature oligodendrocytes in the corpus callosum while it does not affect the number of OPCs. Taking together, our results suggest that diosgenin promotes the differentiation of OPC into mature oligodendrocyte through an ER-mediated ERK1/2 activation pathway to accelerate remyelination, which implicates a novel therapeutic usage of this steroidal natural product in demyelinating diseases such as multiple sclerosis (MS).

  12. RNAi‑mediated knockdown of PRL‑3 inhibits cell invasion and downregulates ERK 1/2 expression in the human gastric cancer cell line, SGC‑7901.

    PubMed

    Cao, Yi; Tu, Yi; Mei, Jinhong; Li, Zhengrong; Jie, Zhigang; Xu, Shan; Xu, Linlin; Wang, Shanshan; Xiong, Yifeng

    2013-06-01

    The deregulated expression of members of the phophatase of regenerating liver (PRL) family is important in the metastatic progression of multiple human cancers; however, the underlying mechanisms are not well understood. Previous studies have demonstrated that PRLs are able to enhance the activation of extracellular signal‑regulated kinase 1/2 (ERK 1/2) in cancer cells, which may contribute to tumor metastasis. However, the effect of PRL‑3 activation in gastric cancer (GC) remains unclear. The present study aimed to investigate whether the downregulation of PRL‑3 by small interfering RNA (siRNA) was able to inhibit cell motility and affect ERK 1/2 expression in human GC. The results demonstrated that the downregulation of PRL‑3 expression by siRNA in human GC cells significantly inhibited cell invasion and migration in vitro; accordingly, inhibition of PRL‑3 also prevented ERK1/2 protein and mRNA expression, and reduced the mRNA level of matrix metalloproteinase‑7 (MMP‑7), the downstream target of ERK 1/2 signaling. Our data demonstrated that RNAi‑mediated downregulation of PRL‑3 expression leads to potent antitumor activity in human GC. Furthermore, ERK 1/2 and MMP‑7 may contribute to the carcinogenesis and development of human GC in combination with PRL‑3.

  13. Down-regulation of tumor endothelial marker 8 suppresses cell proliferation mediated by ERK1/2 activity

    PubMed Central

    Cao, Chuangjie; Wang, Zhuo; Huang, Leilei; Bai, Lihong; Wang, Yuefeng; Liang, Yingjie; Dou, Chengyun; Wang, Liantang

    2016-01-01

    Tumor endothelial marker 8 (TEM8) was recently suggested as a putative anti-tumor target in several types of human cancer based on its selective overexpression in tumor versus normal endothelial cells. The objective of this study was to detect the potential functions of TEM8 in osteosarcoma. Overall, TEM8 was mainly located in cytoplasm and was up-regulated in osteosarcoma compared to benign bone lesions and adjacent non tumor tissue (ANT). High TEM8 expression group had a significant lower overall survival rate than that in the low TEM8 expression group. TEM8 knock-down by siRNA or shRNA results in significant reduction of osteosarcoma cell growth and proliferation both in vitro and in vivo. Ablation of TEM8 led to increasing of p21 and p27 and suppression of cyclin D1 mediated by Erk1/2 activity. These findings suggest that down-regulation of TEM8 play an important role in the inhibition of tumorigenesis and development of osteosarcoma. PMID:26996335

  14. Neuroprotective effects of Argon are mediated via an ERK-1/2 dependent regulation of heme-oxygenase-1 in retinal ganglion cells.

    PubMed

    Ulbrich, Felix; Kaufmann, Kai B; Coburn, Mark; Lagrèze, Wolf Alexander; Roesslein, Martin; Biermann, Julia; Buerkle, Hartmut; Loop, Torsten; Goebel, Ulrich

    2015-08-01

    Retinal ischemia and reperfusion injuries (R-IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti-apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK-1/2 dependent regulation of heat-shock proteins. Inhalation of Argon (75 Vol%) was performed after R-IRI on the rats' left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat-shock proteins -70, -90 and heme-oxygenase-1, mitogen-activated protein kinases (p38, JNK, ERK-1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova. Argon significantly reduced the R-IRI-affected heat-shock protein expression (p < 0.05). While Argon significantly induced ERK-1/2 expression (p < 0.001), inhibition of ERK-1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme-oxygenase-1 (p < 0.05). R-IRI-induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK-1/2 activation in Müller cells. We conclude, that Argon treatment protects R-IRI-induced apoptotic loss of RGC via an ERK-1/2 dependent regulation of heme-oxygenase-1. We proposed the following possible mechanism for Argon-mediated neuroprotection: Argon exerts its protective effects via an induction of an ERK with subsequent suppression of the heat shock response. In conclusion, ischemia and reperfusion injuries and subsequent neuronal apoptosis are attenuated. These novel findings may open up new opportunities for Argon as a therapeutic option, especially since Argon is not toxic.

  15. Dietary salt modulates the sodium chloride cotransporter expression likely through an aldosterone-mediated WNK4-ERK1/2 signaling pathway.

    PubMed

    Lai, Lingyun; Feng, Xiuyan; Liu, Defeng; Chen, Jing; Zhang, Yiqian; Niu, Bowen; Gu, Yong; Cai, Hui

    2012-03-01

    WNK is a serine/threonine kinase. Mutation in WNK1 or WNK4 kinase results in pseudohypoaldosteronism type II (PHA II) featuring hypertension, hyperkalemia and metabolic acidosis. Sodium chloride cotransporter (NCC) is known to be regulated by phosphorylation and trafficking. Dietary salt and hormonal stimulation, such as aldosterone, also affect the regulation of NCC. We have previously reported that WNK4 inhibits NCC protein expression. To determine whether dietary salt affects NCC abundance through WNK4-mediated mechanism, we investigated the effects of dietary salt change with or without aldosterone infusion (1 mg/kg/day) on NCC and WNK4 expression in rats. We found that high-salt (HS, 4% NaCl) diet significantly inhibits NCC mRNA expression and protein abundance while enhancing WNK4 mRNA and protein expression, whereas low-salt (LS, 0.07% NaCl) diet increases NCC mRNA expression and protein abundance while reducing WNK4 expression. We also found that aldosterone infusion in HS-fed rats increases NCC mRNA expression and protein abundance, but decreases WNK4 expression. Administration with spironolactone (0.1 g/kg/day) in LS-fed rats decreases NCC mRNA expression and protein abundance while increasing WNK4 expression. We further showed that ERK1/2 phosphorylation was increased in HS-fed rats, but decreased in LS-fed rats. In HEK293 cells, over-expressed WNK4 increases ERK1/2 phosphorylation, whereas knockdown of WNK4 expression decreases ERK1/2 phosphorylation. Aldosterone treatment for 3 h decreases ERK1/2 phosphorylation. These data suggest that dietary salt change affects NCC protein abundance in an aldosterone-dependent mechanism likely via the WNK4-ERK1/2-mediated pathway.

  16. Adenosine A{sub 2A} receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    SciTech Connect

    Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

    2013-05-10

    Highlights: •A{sub 2A} receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A{sub 2A} receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A{sub 2A} receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A{sub 2A} receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A{sub 2A} receptor. A{sub 2A} receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A{sub 2A} receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A{sub 2A} receptor-overexpressing HLMVECs. Adenoviral-mediated A{sub 2A} receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A{sub 2A} receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A{sub 2A} receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A{sub 2A} receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A{sub 2A}-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A{sub 2A} receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

  17. Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/β-arrestin activation of ERK1/2 and Src kinase.

    PubMed

    Mahavadi, Sunila; Sriwai, Wimolpak; Huang, Jiean; Grider, John R; Murthy, Karnam S

    2014-03-01

    We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two Gβγ-dependent pathways (PLC-β3 and phosphatidylinositol 3-kinase/integrin-linked kinase). Here we show that the anticipated Gβγ-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via Gαi, signaling resulted from Gβγ-independent phosphorylation of CB1 receptors by GRK5, recruitment of β-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by pertussis toxin (PTx) or Gi minigene nor expression of a Gβγ-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing β-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated phosphoinositide hydrolysis or enhance Gαq:RGS4 association and inactivation of Gαq. Activation of Src kinase by anandamide enhanced both myosin phosphatase RhoA-interacting protein (M-RIP):RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity, leading to increase of myosin light chain (MLC) phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage Gβγ but signaled via GRK5/β-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of Gαq by RGS4, and Src kinase enhanced MLC phosphatase activity, leading to inhibition of ACh-stimulated contraction.

  18. Effect of ERK1/2 Signaling Pathway in Electro-Acupuncture – Mediated Up-Regulation of Heme Oxygenase-1 in Lungs of Rabbits with Endotoxic Shock

    PubMed Central

    Zhang, Yuan; Yu, Jian-bo; Luo, Xiao-qing; Gong, Li-rong; Wang, Man; Cao, Xin-shun; Dong, Shu-an; Yan, Yu-miao; Kwon, Yihyun; He, Jia

    2014-01-01

    Background The anti-oxidative and anti-inflammatory activities of electro-acupuncture (EA), a traditional clinical method, are widely accepted, but its mechanisms are not yet well defined. In this study, we investigated the role of extracellular signal-regulated kinases1/2 (ERK1/2) pathways on electro-acupuncture – mediated up-regulation of heme oxygenase-1 (HO-1) in rabbit lungs injured by LPS-induced endotoxic shock. Material/Methods Seventy rabbits were randomly divided into 7 groups: group C, group M, group D, group SEAM, group EAM, group EAMPD, and group PD98059. Male New England white rabbits were given EA treatment on both sides once a day on days 1–5, and then received LPS to replicate the experimental model of injured lung induced by endotoxic shock. Then, they were killed by exsanguination at 6 h after LPS administration. The blood samples were collected for serum examination, and the lungs were removed for pathology examination, determination of wet-to-dry weight ratio, MDA content, SOD activity, serum tumor necrosis factor-α, determination of HO-1 protein and mRNA expression, and determination of ERK1/2 protein. Results The results revealed that after EA treatment, expression of HO-1and ERK1/2 was slightly increased compared to those in other groups, accompanied with less severe lung injury as indicated by lower index of lung injury score, lower wet-to-dry weight ratio, MDA content, and serum tumor necrosis factor-α levels, and greater SOD activity (p<0.05 for all). After pretreatment with ERK1/2 inhibitor PD98059, the effect of EA treatment and expression of HO-1 were suppressed (p<0.05 for all). Conclusions After electro-acupuncture stimulation at ST36 and BL13, severe lung injury during endotoxic shock was attenuated. The mechanism may be through up-regulation of HO-1, mediated by the signal transductions of ERK1/2 pathways. Thus, the regulation of ERK1/2 pathways via electro-acupuncture may be a therapeutic strategy for endotoxic shock. PMID

  19. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    SciTech Connect

    Song, Shasha; Wang, Shuang; Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin; Zhu, Daling

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  20. Aquaporin-8 mediates human esophageal cancer Eca-109 cell migration via the EGFR-Erk1/2 pathway

    PubMed Central

    Chang, Heng; Shi, Yong-Hua; Talaf, Tuo-Kan; Lin, Chen

    2014-01-01

    Abnormal expression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases1/2 (ERK1/2) are associated with tumorigenesis and cancer progression and may upregulate AQPs expression. In this study, we investigated acquaporin-8 expression and signaling via epidermal growth factor receptor-extracellular signal-regulated kinases1/2 in human esophageal cancer Eca-109 cells by western blot, immunofluorescence and wound healing (scratch) assays. Our results showed that epidermal growth factor (EGF) induced both Eca-109 migration and AQP8 expression. Wound healing results showed that cell migration was increased by 1.23-1.10-fold at 24 h and 48 h after EGF treatment. AQP8 expression was significantly increased (1.19-fold) at 48 h after EGF treatment in Eca-109. The EGFR kinase inhibitor, PD153035, blocked EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 3.65-fold (EGF-treated) to 0.55-fold (PD153035-treated) in Eca-109. Furthermore, the MEK [MAPK (mitogen-activated protein kinase)/Erk1/2]/Erk1/2 inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 3.92-fold (EGF-treated) to 1.38-fold (U0126-treated) in Eca-109. In conclusions, EGF induces AQP8 expression and cell migration in Eca-109 cells via the EGFR/Erk1/2 signal transduction pathway. PMID:25550802

  1. Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS.

    PubMed

    Haupaix, Nicolas; Abitua, Philip B; Sirour, Cathy; Yasuo, Hitoyoshi; Levine, Michael; Hudson, Clare

    2014-10-01

    Recent evidence suggests that ascidian pigment cells are related to neural crest-derived melanocytes of vertebrates. Using live-imaging, we determine a revised cell lineage of the pigment cells in Ciona intestinalis embryos. The neural precursors undergo successive rounds of anterior-posterior (A-P) oriented cell divisions, starting at the blastula 64-cell stage. A previously unrecognized fourth A-P oriented cell division in the pigment cell lineage leads to the generation of the post-mitotic pigment cell precursors. We provide evidence that MEK/ERK signals are required for pigment cell specification until approximately 30min after the final cell division has taken place. Following each of the four A-P oriented cell divisions, ERK1/2 is differentially activated in the posterior sister cells, into which the pigment cell lineage segregates. Eph/ephrin signals are critical during the third A-P oriented cell division to spatially restrict ERK1/2 activation to the posterior daughter cell. Targeted inhibition of Eph/ephrin signals results in, at neurula stages, anterior expansion of both ERK1/2 activation and a pigment cell lineage marker and subsequently, at larval stages, supernumerary pigment cells. We discuss the implications of these findings with respect to the evolution of the vertebrate neural crest.

  2. A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes.

    PubMed

    Song, Huanghe; Liang, Wenwei; Xu, Shun; Li, Zeng; Chen, Zhefeng; Cui, Weiding; Zhou, Jinchun; Wang, Qing; Liu, Feng; Fan, Weimin

    2016-07-01

    In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress on chondrocytes. Integrin β1-mediated ERK1/2 activation was proven to be indispensable in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. However, other signal proteins responsible for the mitogenesis of chondrocytes under periodic mechanical stress remain incompletely understood. In the current investigation, we probed the roles of integrin-linked kinase (ILK) signaling in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. We found that upon periodic mechanical stress induction, ILK activity increased significantly. Depletion of ILK with targeted shRNA strongly inhibited periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. In addition, pretreatment with a blocking antibody against integrin β1 resulted in a remarkable decrease in ILK activity in cells exposed to periodic mechanical stress. Furthermore, inhibition of ILK with its target shRNA significantly suppressed ERK1/2 activation in relation to periodic mechanical stress. Based on the above results, we identified ILK as a crucial regulator involved in the integrin β1-ERK1/2 signal cascade responsible for periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis.

  3. STC1 induction by PACAP is mediated through cAMP and ERK1/2 but not PKA in cultured cortical neurons

    PubMed Central

    Holighaus, Yvonne; Weihe, Eberhard; Eiden, Lee E.

    2011-01-01

    The neuroprotective actions of PACAP (pituitary adenylate cyclase-activating polypeptide) in vitro and in vivo suggest that activation of its cognate G protein-coupled receptor PAC1 or downstream signaling molecules, and thus activation of PACAP target genes, could be of therapeutic benefit. Here we show, that cultured rat cortical neurons predominantly expressed the PAC1hop and null variants, activation of which resulted in elevation of the two second messengers cAMP and Ca2+ and expression of the putative neuroprotectant stanniocalcin 1 (STC1). PACAP signaling to the STC1 gene proceeded through the extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not through the cAMP dependent protein kinase (PKA), and was mimicked by the adenylate cyclase activator forskolin. PACAP- and forskolin-mediated activation of ERK1/2 occurred through cAMP, but not PKA. These results suggest that STC1 gene induction proceeds through cAMP and ERK1/2, independently of PKA, the canonical cAMP effector. In contrast, PACAP signaling to the BDNF gene proceeded through PKA, suggesting that two different neuroprotective cAMP pathways co-exist in differentiated cortical neurons. The selective activation of a potentially neuroprotective cAMP dependent pathway different from the canonical cAMP pathway used in many physiological processes, such as memory storage, has implications for pharmacological activation of neuroprotection in vivo. PMID:21975601

  4. Gain-of-Function Mutations in the Toll-Like Receptor Pathway: TPL2-Mediated ERK1/ERK2 MAPK Activation, a Path to Tumorigenesis in Lymphoid Neoplasms?

    PubMed Central

    Rousseau, Simon; Martel, Guy

    2016-01-01

    Lymphoid neoplasms form a family of cancers affecting B-cells, T-cells, and NK cells. The Toll-Like Receptor (TLR) signaling adapter molecule MYD88 is the most frequently mutated gene in these neoplasms. This signaling adaptor relays signals from TLRs to downstream effector pathways such as the Nuclear Factor kappa B (NFκB) and Mitogen Activated Protein Kinase (MAPK) pathways to regulate innate immune responses. Gain-of-function mutations such as MYD88[L265P] activate downstream signaling pathways in absence of cognate ligands for TLRs, resulting in increased cellular proliferation and survival. This article reports an analysis of non-synonymous somatic mutations found in the TLR signaling network in lymphoid neoplasms. In accordance with previous reports, mutations map to MYD88 pro-inflammatory signaling and not TRIF-mediated Type I IFN production. Interestingly, the analysis of somatic mutations found downstream of the core TLR-signaling network uncovered a strong association with the ERK1/2 MAPK cascade. In support of this analysis, heterologous expression of MYD88[L265P] in HEK293 cells led to ERK1/2 MAPK phosphorylation in addition to NFκB activation. Moreover, this activation is dependent on the protein kinase Tumor Promoting Locus 2 (TPL2), activated downstream of the IKK complex. Activation of ERK1/2 would then lead to activation, amongst others, of MYC and hnRNPA1, two proteins previously shown to contribute to tumor formation in lymphoid neoplasms. Taken together, this analysis suggests that TLR-mediated ERK1/2 activation via TPL2 may be a novel path to tumorigenesis. Therefore, the hypothesis proposed is that inhibition of ERK1/2 MAPK activation would prevent tumor growth downstream of MYD88[L265]. It will be interesting to test whether pharmacological inhibitors of this pathway show efficacy in primary tumor cells derived from hematologic malignancies such as Waldenstrom's Macroglobulinemia, where the majority of the cells carry the MYD88[L265P

  5. Urocortin-1 Mediated Cardioprotection Involves XIAP and CD40-Ligand Recovery: Role of EPAC2 and ERK1/2

    PubMed Central

    Ordóñez, Antonio; Smani, Tarik

    2016-01-01

    Aims Urocortin-1 (Ucn-1) is an endogenous peptide that protects heart from ischemia and reperfusion (I/R) injuries. Ucn-1 is known to prevent cardiac cell death, but its role in the transcription of specific genes related to survival signaling pathway has not been fully defined. The aim of this study was to investigate the molecular signaling implicated in the improvement of cardiac myocytes survival induced by Ucn-1. Methods and Results Ucn-1 administration before ischemia and at the onset of reperfusion, in rat hearts perfused in Langendorff system, fully recovered heart contractility and other hemodynamic parameters. Ucn-1 enhanced cell viability and decreased lactate dehydrogenase (LDH) release in adult cardiac myocytes subjected to simulated I/R. Annexin V-FITC/PI staining indicated that Ucn-1 promoted cell survival and decreased cell necrosis through Epac2 (exchange protein directly activated by cAMP) and ERK1/2 (extracellular signal–regulated kinases 1/2) activation. We determined that Ucn-1 shifted cell death from necrosis to apoptosis and activated caspases 9 and 3/7. Furthermore, mini-array, RT-qPCR and protein analyses of apoptotic genes showed that Ucn-1 upregulated the expression of CD40lg, Xiap and BAD in cells undergoing I/R, involving Epac2 and ERK1/2 activation. Conclusions Our data indicate that Ucn-1 efficiently protected hearts from I/R damage by increasing the cell survival and stimulated apoptotic genes, CD40lg, Xiap and BAD, overexpression through the activation of Epac2 and ERK1/2. PMID:26840743

  6. Insulin-like growth factors inhibit dendritic cell-mediated anti-tumor immunity through regulating ERK1/2 phosphorylation and p38 dephosphorylation.

    PubMed

    Huang, Ching-Ting; Chang, Ming-Cheng; Chen, Yu-Li; Chen, Tsung-Ching; Chen, Chi-An; Cheng, Wen-Fang

    2015-04-01

    Insulin-like growth factors (IGFs) can promote tumorigenesis via inhibiting the apoptosis of cancer cells. The relationship between IGFs and dendritic cell (DC)-mediated immunity were investigated. Advanced-stage ovarian carcinoma patients were first evaluated to show higher IGF-1 and IGF-2 concentrations in their ascites than early-stage patients. IGFs could suppress DCs' maturation, antigen presenting abilities, and the ability to activate antigen-specific CD8(+) T cell. IGF-treated DCs also secreted higher concentrations of IL-10 and TNF-α. IGF-treated DCs showed decreased ERK1/2 phosphorylation and reduced p38 dephosphorylation. The percentages of matured DCs in the ascites were significantly lower in the IGF-1 or IGF-2 highly-expressing WF-3 tumor-bearing mice. The IGF1R inhibitor - NVP-AEW541, could block the effects of IGFs to rescue DCs' maturation and to restore DC-mediated antigen-specific immunity through enhancing ERK1/2 phosphorylation and p38 dephosphorylation. IGFs can inhibit DC-mediated anti-tumor immunity through suppressing maturation and function and the IGF1R inhibitor could restore the DC-mediated anti-tumor immunity. Blockade of IGFs could be a potential strategy for cancer immunotherapy.

  7. Human Amnion-Derived Mesenchymal Stem Cells Protect Human Bone Marrow Mesenchymal Stem Cells against Oxidative Stress-Mediated Dysfunction via ERK1/2 MAPK Signaling

    PubMed Central

    Wang, Yuli; Ma, Junchi; Du, Yifei; Miao, Jing; Chen, Ning

    2016-01-01

    Epidemiological evidence suggests that bone is especially sensitive to oxidative stress, causing bone loss in the elderly. Previous studies indicated that human amnion-derived mesenchymal stem cells (HAMSCs), obtained from human amniotic membranes, exerted osteoprotective effects in vivo. However, the potential of HAMSCs as seed cells against oxidative stress-mediated dysfunction is unknown. In this study, we systemically investigated their antioxidative and osteogenic effects in vitro. Here, we demonstrated that HAMSCs signi cantly promoted the proliferation and osteoblastic differentiation of H2O2-induced human bone marrow mesenchymal stem cells (HBMSCs), and down-regulated the reactive oxygen species (ROS) level. Further, our results suggest that activation of the ERK1/2 MAPK signal transduction pathway is essential for both HAMSCs-mediated osteogenic and protective effects against oxidative stress-induced dysfunction in HBMSCs. U0126, a highly selective inhibitor of extracellular ERK1/2 MAPK signaling, significantly suppressed the antioxidative and osteogenic effects in HAMSCs. In conclusion, by modulating HBMSCs, HAMSCs show a strong potential in treating oxidative stress- mediated bone deficiency. PMID:26743906

  8. Anti-adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways.

    PubMed

    Choi, Jae Sue; Kim, Ji-Hye; Ali, Md Yousof; Jung, Hee Jin; Min, Byung-Sun; Choi, Ran Joo; Kim, Gun-Do; Jung, Hyun Ah

    2015-12-01

    It has been reported that alkaloids derived from Coptis chinensis exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the inhibitory role of epiberberine in the early stages of 3T3-L1 adipocyte differentiation is uncharacterized. Here, we show that epiberberine had inhibitory effects on adipocyte differentiation and significantly decreased lipid accumulation by downregulating an adipocyte-specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). Furthermore, we observed that epiberberine markedly suppressed the differentiation-mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly inhibited by treatment with epiberberine during adipogenesis. These results indicate that the anti-adipogenic mechanism of epiberberine is associated with inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of adipogenesis, such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the anti-adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 adipocyte differentiation. Moreover, the anti-adipogenic effects of epiberberine were not accompanied by modulation of β-catenin.

  9. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    PubMed

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  10. Growth Differentiation Factor-8 Decreases StAR Expression Through ALK5-Mediated Smad3 and ERK1/2 Signaling Pathways in Luteinized Human Granulosa Cells.

    PubMed

    Fang, Lanlan; Chang, Hsun-Ming; Cheng, Jung-Chien; Yu, Yiping; Leung, Peter C K; Sun, Ying-Pu

    2015-12-01

    Growth differentiation factor-8 (GDF-8) has been recently shown to be expressed in human granulosa cells, and the mature form of GDF-8 protein can be detected in the follicular fluid. However, the biological function and significance of this growth factor in the human ovary remains to be determined. Here, we investigated the effects of GDF-8 on steroidogenic enzyme expression and the potential mechanisms of action in luteinized human granulosa cells. We demonstrated that treatment with GDF-8 did not affect the mRNA levels of P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase, whereas it significantly down-regulated steroidogenic acute regulatory protein (StAR) expression and decreased progesterone production. The suppressive effect of GDF-8 on StAR expression was abolished by the inhibition of the TGF-β type I receptor. In addition, treatment with GDF-8 activated both Smad2/3 and ERK1/2 signaling pathways. Furthermore, knockdown of activin receptor-like kinase 5 reversed the effects of GDF-8 on Smad2/3 phosphorylation and StAR expression. The inhibition of Smad3 or ERK1/2 signaling pathways attenuated the GDF-8-induced down-regulation of StAR and production of progesterone. Interestingly, the concentrations of GDF-8 were negatively correlated with those of progesterone in human follicular fluid. These results indicate a novel autocrine function of GDF-8 to down-regulate StAR expression and decrease progesterone production in luteinized human granulosa cells, most likely through activin receptor-like kinase 5-mediated Smad3 and ERK1/2 signaling pathways. Our findings suggest that granulosa cells might play a critical role in the regulation of progesterone production to prevent premature luteinization during the final stage of folliculogenesis.

  11. Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI{sub 3}K and mTOR

    SciTech Connect

    Wang, Yanling; Sato, Masaaki; Guo, Yuan; Bengtsson, Tore; Nedergaard, Jan

    2014-10-15

    The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes during the process of tissue recruitment. In a primary culture system, cAMP mediates these adrenergic effects. In the present study, we demonstrated that, in contrast to other systems where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors, especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2 family of MAPK, the PI{sub 3}K system and the mTOR complexes were all activated by cAMP, but these activations were not necessary for cAMP-induced cell proliferation; a protein kinase C isoform may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally acknowledged cellular mediators for induction of cell proliferation are not involved in this process in the brown preadipocyte system; this conclusion may be of relevance both for examination of mechanisms for induction of brown adipose tissue recruitment but also for understanding the mechanism behind e.g. certain endocrine neoplasias. - Highlights: • cAMP can mimick norepinephrine-induced proliferation of brown preadipocytes. • The cAMP-induced proliferation can occur in the absence of serum, of any other growth factors, and of insulin. • Erk1/2, PI{sub 3}K and mTOR are cAMP activated but not involved in induction of proliferation. • A Protein Kinase C member may be in the signalling cascade. • This pathway analysis may also be of importance for certain endocrine hyper- and neoplasias.

  12. Betulinic acid exerts anti-hepatitis C virus activity via the suppression of NF-κB- and MAPK-ERK1/2-mediated COX-2 expression

    PubMed Central

    Lin, Chun-Kuang; Tseng, Chin-Kai; Chen, Kai-Hsun; Wu, Shih-Hsiung; Liaw, Chih-Chuang; Lee, Jin-Ching

    2015-01-01

    Background and Purpose This study was designed to evaluate the effect of betulinic acid (BA), extracted from Avicennia marina, on the replication of hepatitis C virus (HCV) and to investigate the mechanism of this BA-mediated anti-HCV activity. Experimental Approach HCV replicon and infectious systems were used to evaluate the anti-HCV activity of BA. Exogenous COX-2 or knock-down of COX-2 expression was used to investigate the role of COX-2 in the anti-HCV activity of BA. The effects of BA on the phosphorylation of NF-κB and on kinases in the MAPK signalling pathway were determined. The anti-HCV activity of BA in combination with other HCV inhibitors was also determined to assess its use as an anti-HCV supplement. Key Results BA inhibited HCV replication in both Ava5 replicon cells and in a cell culture-derived infectious HCV particle system. Treatment with a combination of BA and IFN-α, the protease inhibitor telaprevir or the NS5B polymerase inhibitor sofosbuvir resulted in the synergistic suppression of HCV RNA replication. Exogenous overexpression of COX-2 gradually attenuated the inhibitory effect of BA on HCV replication, suggesting that BA reduces HCV replication by suppressing the expression of COX-2. In particular, BA down-regulated HCV-induced COX-2 expression by reducing the phosphorylation of NF-κB and ERK1/2 of the MAPK signalling pathway. Conclusions and Implications BA inhibits HCV replication by suppressing the NF-κB- and ERK1/2-mediated COX-2 pathway and may serve as a promising compound for drug development or as a potential supplement for use in the treatment of HCV-infected patients. PMID:26102077

  13. Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway.

    PubMed

    Xu, Xiaohong; Lu, Yang; Zhang, Guangxia; Chen, Lei; Tian, Dong; Shen, Xiuying; Yang, Yanling; Dong, Fanni

    2014-02-01

    Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30min. The purpose of the present study was further to investigate the effects of BPA for 24h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5d in vitro, the hippocampal neurons from 24h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100nM) or 17β-estradiol (17β-E2, 10nM) for 24h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen-activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17β-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear

  14. All trans-retinoic acid analogs promote cancer cell apoptosis through non-genomic Crabp1 mediating ERK1/2 phosphorylation

    PubMed Central

    Persaud, Shawna D.; Park, Sung Wook; Ishigami-Yuasa, Mari; Koyano-Nakagawa, Naoko; Kagechika, Hiroyuki; Wei, Li-Na

    2016-01-01

    All trans retinoic acid (atRA) is one of the most potent therapeutic agents, but extensive toxicity caused by nuclear RA receptors (RARs) limits its clinical application in treating cancer. AtRA also exerts non-genomic activities for which the mechanism remains poorly understood. We determine that cellular retinoic acid binding protein 1 (Crabp1) mediates the non-genomic activity of atRA, and identify two compounds as the ligands of Crabp1 to rapidly and RAR-independently activate extracellular signal regulated kinase 1/2 (ERK1/2). Non-canonically activated ERK activates protein phosphatase 2A (PP2A) and lengthens cell cycle duration in embryonic stem cells (ESC). This is abolished in Crabp1-null ESCs. Re-expressing Crabp1 in Crabp1-negative cancer cells also sensitizes their apoptotic induction by atRA. This study reveals a physiological relevance of the non-genomic action of atRA, mediated by Crabp1, in modulating cell cycle progression and apoptosis induction, and provides a new cancer therapeutic strategy whereby compounds specifically targeting Crabp1 can modulate cell cycle and cancer cell apoptosis in a RAR-independent fashion, thereby avoiding atRA’s toxicity caused by its genomic effects. PMID:26935534

  15. PKA, Rap1, ERK1/2, and p90RSK mediate PGE2 and EP4 signaling in neonatal ventricular myocytes.

    PubMed

    He, Quan; Harding, Pamela; LaPointe, Margot C

    2010-01-01

    We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE(2) production reduces hypertrophy after myocardial infarction in mice and 2) PGE(2) acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE(2) activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE(2) activation of ERK1/2. PGE(2) activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE(2) regulation of gene expression. PGE(2) stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE(2) stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE(2) regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos.

  16. OCT4 mediates FSH-induced epithelial-mesenchymal transition and invasion through the ERK1/2 signaling pathway in epithelial ovarian cancer.

    PubMed

    Liu, Lei; Zhang, Jing; Fang, Chi; Zhang, Zhenbo; Feng, Youji; Xi, Xiaowei

    2015-06-01

    Our previous study showed that Octamer-binding transcription factor 4 (OCT4) expression was upregulated and significantly associated with histological grade through the analysis of OCT4 expression in 159 ovarian cancer tissue samples, and OCT4 mediated follicle-stimulating hormone (FSH)-induced anti-apoptosis in epithelial ovarian cancer. Nevertheless, whether OCT4 participates in FSH-induced invasion in ovarian cancer is still unknown. Therefore, the present study aimed to define whether FSH-induced ovarian cancer invasion is mediated by OCT4. In present study, we showed that FSH induced not only the epithelial-mesenchymal transition (EMT) and invasive phenotype but also the upregulation of OCT4 expression in a dose- and time-dependent manner in epithelial ovarian cancer cells. In addition, the expression of FSH receptor (FSHR) was upregulated by FSH induction, and knockdown of FSHR inhibited FSH-stimulated OCT4 expression. ERK1/2 signaling pathway participated in the enhanced expression of OCT4 and Snail induced by FSH. We further showed that the activated expression of Snail and N-cadherin, the suppressed expression of E-cadherin and the morphological change of the cells stimulated by FSH were blocked by OCT4-specific small interfering RNA. Moreover, our results showed that OCT4 mediated the increase in invasive capacity induced by FSH in ovarian cancer cells. Taken together, our work reveals that OCT4 is an essential mediator in FSH-induced EMT and invasion in epithelial ovarian cancer and may act as a potential therapeutic target.

  17. ET-1-induced growth promoting responses involving ERK1/2 and PKB signaling and Egr-1 expression are mediated by Ca2+/CaM-dependent protein kinase-II in vascular smooth muscle cells.

    PubMed

    Bouallegue, Ali; Simo Cheyou, Estelle R; Anand-Srivastava, Madhu B; Srivastava, Ashok K

    2013-12-01

    Endothelin-1 (ET-1), a potent vasoactive peptide with a pathogenic role in vascular diseases, has been shown to induce the activation of ERK1/2, PKB and the expression of a transcriptional regulator, the early growth response 1 (Egr-1), key mediators of hypertrophic and proliferative responses in vascular smooth muscle cells (VSMC). We have demonstrated earlier that ET-1 requires H2O2 generation to activate these signaling pathways and Ca2+, calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII), play a critical role to trigger H2O2-induced effects in VSMC. However, an involvement of CaMKII in mediating ET-1-induced responses in VSMC remains unknown. Therefore, by utilizing pharmacological inhibitors of CaM, CaMKII, a CaMKII inhibitor peptide and CaMKII knockdown techniques, we have investigated the contribution of CaM and CaMKII in ET-1-induced ERK1/2 and PKB signaling, Egr-1 expression and hypertrophic and proliferative responses in VSMC. W-7 and calmidazolium, antagonists of CaM, as well as KN-93, an inhibitor of CaMKII activity, attenuated ET-1-induced ERK1/2 and PKB phosphorylation. In addition, transfection of VSMC with a CaMKII inhibitory peptide suppressed ET-1-evoked ERK1/2 and PKB phosphorylation. Similarly, siRNA-mediated CaMKII silencing reduced ET-1-produced ERK1/2 and PKB phosphorylation. CaM and CaMKII blockade also significantly lowered the ET-1-induced protein and DNA synthesis as well as Egr-1 expression. These findings demonstrate that CaMKII plays a critical role in ET-1-induced growth promoting signaling pathways as well as hypertrophic and proliferative responses in VSMC.

  18. Involvement of IGF-1 and MEOX2 in PI3K/Akt1/2 and ERK1/2 pathways mediated proliferation and differentiation of perivascular adipocytes

    SciTech Connect

    Liu, Ping; Kong, Feng; Wang, Jue; Lu, Qinghua; Xu, Haijia; Qi, Tonggang; Meng, Juan

    2015-02-01

    Perivascular adipocyte (PVAC) proliferation and differentiation were closely involved in cardiovascular disease. We aimed to investigate whether phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways enhance PVAC functions activated by insulin-like growth factor 1(IGF-1) and suppressed by mesenchyme homeobox 2 (MEOX2). In this study, PVACs from primary culture were cultured and induced to differentiate. Cell viability assays demonstrated that IGF-1 promoted PVAC proliferation and differentiation. However MEOX2 counteracted these IGF-1-mediated actions. Flow Cytometry revealed that IGF-1 increased S phase cells and decreased apoptosis; however, MEOX2 decreased S phase cells, increased G0–G1 phase cells, and promoted apoptosis. During PVAC proliferation and differentiation, IGF-1 activated PI3K/Akt1/2 and ERK1/2 signaling pathways, upregulated the expression of these signaling proteins and FAS, and increased PVAC lipid content. In contrast, MEOX2 constrained the phosphorylation of ERK1/2 and Akt1/2 protein, down-regulated these signaling molecules and FAS, and decreased PVAC lipid content. Instead, MEOX2 knockdown enhanced the ERK1/2 and Akt1/2 phosphorylation, augmented the expression of these signaling molecules and FAS, and increased PVAC lipid content. Our findings suggested that PI3K/Akt1/2 and ERK1/2 activation mediated by IGF-1 is essential for PVAC proliferation and differentiation, and MEOX2 is a promising therapeutic gene to intervene in the signaling pathways and inhibit PVAC functions. - Highlights: • IGF-1 activated PI3K/Akt2 and ERK1/2 pathways to mediate PVAC proliferation and differentiation. • The expression of ERK1, ERK 2, PI3K, Akt1 and Akt2 showed different change trends between PVAC proliferation and differentiation. • MEOX2 effectively expressed in PVAC, increased early and late cellular apoptosis, and inhibited its proliferation. • MEOX2 depressed PVAC differentiation and FAS expression

  19. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals.

    PubMed

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-07-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  20. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals

    PubMed Central

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-01-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  1. Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals.

    PubMed

    Chu, Chun-Hsien; Wang, Shijun; Li, Chia-Ling; Chen, Shih-Heng; Hu, Chih-Fen; Chung, Yi-Lun; Chen, Shiou-Lan; Wang, Qingshan; Lu, Ru-Band; Gao, Hui-Ming; Hong, Jau-Shyong

    2016-07-01

    Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1β). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important

  2. Rapamycin inhibits BAFF-stimulated cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells.

    PubMed

    Zeng, Qingyu; Zhang, Hai; Qin, Jiamin; Xu, Zhigang; Gui, Lin; Liu, Beibei; Liu, Chunxiao; Xu, Chong; Liu, Wen; Zhang, Shuangquan; Huang, Shile; Chen, Long

    2015-12-01

    B-cell activating factor (BAFF) is involved in not only physiology of normal B cells, but also pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Rapamycin, a lipophilic macrolide antibiotic, has recently shown to be effective in the treatment of human lupus erythematosus. However, how rapamycin inhibits BAFF-stimulated B-cell proliferation and survival has not been fully elucidated. Here, we show that rapamycin inhibited human soluble BAFF (hsBAFF)-induced cell proliferation and survival in normal and B-lymphoid (Raji and Daudi) cells by activation of PP2A and inactivation of Erk1/2. Pretreatment with PD98059, down-regulation of Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2A potentiated rapamycin's suppression of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR-T (mTOR-TE), conferred resistance to rapamycin's effects on PP2A, Erk1/2 and B-cell proliferation/viability, implying mTOR-dependent mechanism involved. The findings indicate that rapamycin inhibits BAFF-stimulated cell proliferation/survival by targeting mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Our data highlight that rapamycin may be exploited for preventing excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

  3. The Neuroprotective Effect of Lithium in cannabinoid Dependence is Mediated through Modulation of Cyclic AMP, ERK1/2 and GSK-3β Phosphorylation in Cerebellar Granular Neurons of Rat.

    PubMed

    Rahimi, Hamid Reza; Ghahremani, Mohammad Hossein; Dehpour, Ahmad Reza; Sharifzadeh, Mohammad; Ejtemaei-Mehr, Shahram; Razmi, Ali; Ostad, Seyed Nasser

    2015-01-01

    Lithium (Li), a glycogen synthase kinase-3β (GSK-3β) inhibitor, has used to attenuate the cannabinoid-induced dependence/withdrawal signs, but molecular mechanisms related to this are unclear. Recent studies indicate the involvement of upstream extracellular signal kinase1/2 (ERK1/2) and downstream GSK-3β pathways in the development of cannabinoid-induced dependence. This is mediated through cannabinoid receptor 1 (CB1) enriched in cerebellar granular neurons (CGNs). Accordingly, the present study aimed to investigate the mechanism of modulatory/neuroprotective effects of Li on a cannabinoid agonist (WIN 55,212-2 (WIN))-induced dependence, through quantitative analysis of some involved proteins such as ERK1/2, GSK-3β and related signaling pathways including their phosphorylated forms; and cAMP level as the other molecular mechanisms leading to dependence, in CGNs model. The CGNs were prepared from 7-day-old Wistar rat pup in a 12-well plate, pretreated with Li (1mM) and an ERK1/2 inhibitor SL327 (SL, 10 µM). The WIN (1 µM) was added 30 minutes prior to treatment and AM251 (AM, 1 µM), as a cannabinoid antagonist was co-treated with WIN. The cAMP level, as an indicator of cannabinoid-induced dependence, was measured by ELISA following forskolin (FSK) stimulation. Western blot analyses determined the phosphorylated forms of ERK1/2 (p-ERK1/2), GSK-3β (p-GSK-3β) as well as their total expressions in various treatment times and doses in CGNs. WIN alone could down regulate the cAMP/p-ERK1/2 cascade compared to AM treatment. However, P-GSK-3β was up-regulated with Li and WIN or with SL and Li pretreatment to AM-induced cellular response, which was the highest 60 minutes after CGNs exposure. Results further suggested the potential role of Li pretreatment to diminish the development of cannabinoid-induced dependence/neuronal injury through possible mechanisms of modulating the cAMP/p-ERK1/2 cascade independent of p-GSK-3β signaling pathway in-vitro.

  4. The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

    SciTech Connect

    Teng, Chih-Chuan; Kuo, Hsing-Chun; Cheng, Ho-Chen; Wang, Ting-Chung; Sze, Chun-I

    2012-08-15

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone) is the major active agent of the alkaloid derivative of Camptotheca acuminata, with multiple pharmacological activities, including anticancer effects and promotion of apoptosis. The mechanism by which CIL-102 inhibits growth remains poorly understood in human astrocytoma cells. Herein, we investigated the molecular mechanisms by which CIL-102 affects the generation of reactive oxygen species (ROS) and cell cycle G2/M arrest in glioma cells. Treatment of U87 cells with 1.0 μM CIL-102 resulted in phosphorylation of extracellular signal-related kinase (ERK1/2), downregulation of cell cycle-related proteins (cyclin A, cyclin B, cyclin D1, and cdk1), and phosphorylation of cdk1Tyr{sup 15} and Cdc25cSer{sup 216}. Furthermore, treatment with the ERK1/2 inhibitor PD98059 abolished CIL-102-induced Cdc25cSer{sup 216} expression and reversed CIL-102-inhibited cdk1 activation. In addition, N-acetyl cysteine (NAC), an ROS scavenger, blocked cell cycle G2/M arrest and phosphorylation of ERK1/2 and Cdc25cSer{sup 216} in U87 cells. CIL-102-mediated ERK1/2 and ROS production, and cell cycle arrest were blocked by treatment with specific inhibitors. In conclusion, we have identified a novel CIL-102-inhibited proliferation in U87 cells by activating the ERK1/2 and Cdc25cSer{sup 216} cell cycle-related proteins and inducing ROS production; this might be a new mechanism in human astrocytoma cells. -- Highlights: ► We show the effects of CIL-102 on the G2/M arrest of human astrocytoma cells. ► ROS and the Ras/ERK1/2 triggering pathways are involved in the CIL-102 treatment. ► CIL-102 induces sustained activation of ERK1/2 and Cdc25c and ROS are required.

  5. The Neuroprotective Effect of Lithium in cannabinoid Dependence is Mediated through Modulation of Cyclic AMP, ERK1/2 and GSK-3β Phosphorylation in Cerebellar Granular Neurons of Rat

    PubMed Central

    Rahimi, Hamid Reza; Ghahremani, Mohammad Hossein; Dehpour, Ahmad Reza; Sharifzadeh, Mohammad; Ejtemaei-Mehr, Shahram; Razmi, Ali; Ostad, Seyed Nasser

    2015-01-01

    Lithium (Li), a glycogen synthase kinase-3β (GSK-3β) inhibitor, has used to attenuate the cannabinoid-induced dependence/withdrawal signs, but molecular mechanisms related to this are unclear. Recent studies indicate the involvement of upstream extracellular signal kinase1/2 (ERK1/2) and downstream GSK-3β pathways in the development of cannabinoid-induced dependence. This is mediated through cannabinoid receptor 1 (CB1) enriched in cerebellar granular neurons (CGNs). Accordingly, the present study aimed to investigate the mechanism of modulatory/neuroprotective effects of Li on a cannabinoid agonist (WIN 55,212-2 (WIN))-induced dependence, through quantitative analysis of some involved proteins such as ERK1/2, GSK-3β and related signaling pathways including their phosphorylated forms; and cAMP level as the other molecular mechanisms leading to dependence, in CGNs model. The CGNs were prepared from 7-day-old Wistar rat pup in a 12-well plate, pretreated with Li (1mM) and an ERK1/2 inhibitor SL327 (SL, 10 µM). The WIN (1 µM) was added 30 minutes prior to treatment and AM251 (AM, 1 µM), as a cannabinoid antagonist was co-treated with WIN. The cAMP level, as an indicator of cannabinoid-induced dependence, was measured by ELISA following forskolin (FSK) stimulation. Western blot analyses determined the phosphorylated forms of ERK1/2 (p-ERK1/2), GSK-3β (p-GSK-3β) as well as their total expressions in various treatment times and doses in CGNs. WIN alone could down regulate the cAMP/p-ERK1/2 cascade compared to AM treatment. However, P-GSK-3β was up-regulated with Li and WIN or with SL and Li pretreatment to AM-induced cellular response, which was the highest 60 minutes after CGNs exposure. Results further suggested the potential role of Li pretreatment to diminish the development of cannabinoid-induced dependence/neuronal injury through possible mechanisms of modulating the cAMP/p-ERK1/2 cascade independent of p-GSK-3β signaling pathway in-vitro. PMID:26664379

  6. TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

    PubMed Central

    L'Abbate, Carolina; Cipriano, Ivone; Pérez-Hurtado, Elizabeth Cristina; Leão, Sylvia Cardoso; Carneiro, Célia Regina Whitaker; Machado, Joel

    2011-01-01

    Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth. PMID:21731758

  7. Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells.

    PubMed

    Tung, Chun-Liang; Jian, Yi-Jun; Syu, Jhan-Jhang; Wang, Tai-Jing; Chang, Po-Yuan; Chen, Chien-Yu; Jian, Yun-Ting; Lin, Yun-Wei

    2015-05-15

    Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. PMID:25662161

  8. Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

    PubMed

    Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

    2015-10-15

    Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2.

  9. Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells

    SciTech Connect

    Tung, Chun-Liang; Jian, Yi-Jun; Syu, Jhan-Jhang; Wang, Tai-Jing; Chang, Po-Yuan; Chen, Chien-Yu; Jian, Yun-Ting; Lin, Yun-Wei

    2015-05-15

    Gefitinib (Iressa{sup R}, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. - Highlights: • Gefitinib treatment decreased XRCC1 mRNA and protein expression in NSCLC cells. • Knocking down XRCC1 expression enhanced the cytotoxic effect of gefitinib. • Gefitinib combined with an Hsp90 inhibitor resulted in synergistically cytotoxicity.

  10. CRHR1 mediates p53 transcription induced by high altitude hypoxia through ERK 1/2 signaling in rat hepatic cells.

    PubMed

    Zhao, Yang; Wang, Ming-Yang; Hao, Ke; Chen, Xue-Qun; Du, Ji-Zeng

    2013-06-01

    We have previously reported that hypoxia activates corticotrophin-releasing hormone (CRH) and the expression of its type-1 receptor (CRHR1) and induces disorders of the brain-endocrine-immune network. p53 is activated by hypoxia and involved in tumorigenesis and apoptosis. Whether CRHR1 regulates p53 transactivation to further influence apoptotic genes remains unclear. Here, we showed that hypoxia at a simulated altitude of 5km or 7km for 8 and 24h increased p53 protein and mRNA, and reduced apoptotic bax and IGFBP3 gene expression while upregulating the cell-arrest gene p21 for 8h in rat liver cells. The upregulation of p53 mRNA and downregulation of bax mRNA induced by hypoxia were blocked by pretreatment with the specific CRHR1 antagonist CP-154,526, but the downregulation of IGFBP3 and upregulation of p21 mRNA were not. Furthermore, CRH stimulated p53 mRNA via the ERK 1/2 pathway in the BRL-3A cell line and this was blocked by the ERK 1/2 antagonist U0126. These data provide novel evidence that the CRHR1-triggered ERK 1/2 pathway is involved in the activation of p53 and suppression of the apoptotic bax gene by hypoxia in rat liver.

  11. CRHR1 mediates p53 transcription induced by high altitude hypoxia through ERK 1/2 signaling in rat hepatic cells.

    PubMed

    Zhao, Yang; Wang, Ming-Yang; Hao, Ke; Chen, Xue-Qun; Du, Ji-Zeng

    2013-06-01

    We have previously reported that hypoxia activates corticotrophin-releasing hormone (CRH) and the expression of its type-1 receptor (CRHR1) and induces disorders of the brain-endocrine-immune network. p53 is activated by hypoxia and involved in tumorigenesis and apoptosis. Whether CRHR1 regulates p53 transactivation to further influence apoptotic genes remains unclear. Here, we showed that hypoxia at a simulated altitude of 5km or 7km for 8 and 24h increased p53 protein and mRNA, and reduced apoptotic bax and IGFBP3 gene expression while upregulating the cell-arrest gene p21 for 8h in rat liver cells. The upregulation of p53 mRNA and downregulation of bax mRNA induced by hypoxia were blocked by pretreatment with the specific CRHR1 antagonist CP-154,526, but the downregulation of IGFBP3 and upregulation of p21 mRNA were not. Furthermore, CRH stimulated p53 mRNA via the ERK 1/2 pathway in the BRL-3A cell line and this was blocked by the ERK 1/2 antagonist U0126. These data provide novel evidence that the CRHR1-triggered ERK 1/2 pathway is involved in the activation of p53 and suppression of the apoptotic bax gene by hypoxia in rat liver. PMID:23538210

  12. Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway.

    PubMed

    Ying, Tsung-Ho; Yang, Shun-Fa; Tsai, Su-Ju; Hsieh, Shu-Ching; Huang, Yi-Chang; Bau, Da-Tian; Hsieh, Yi-Hsien

    2012-02-01

    Fisetin is a naturally occurring flavonoid that has been reported to inhibit the proliferation and to induce apoptotic cell death in several tumor cells. However, the apoptosis-inducing effect of fisetin on tumor cell lines was investigated besides HeLa cells. In this study, we found that fisetin induced apoptosis of HeLa cells in a dose- and time-dependent manner, as evidenced by nuclear staining of 4'-6-Diamidino-2-phenylindole (DAPI), flow cytometry assay, and Annexin-V/PI double-labeling. In addition, fisetin triggered the activations of caspases-3 and -8 and the cleavages of poly (ADP-ribose) polymerase, resulting in apoptosis induction. Moreover, treatment of HeLa cells with fisetin induced a sustained activation of the phosphorylation of ERK1/2, and inhibition of ERK1/2 by PD98059 (MEK1/2 inhibitor) or transfection with the mutant ERK1/2 expression vector significantly abolished the fisetin-induced apoptosis through the activation of caspase-8/-3 pathway. The in vivo xenograft mice experiments revealed that fisetin significantly reduced tumor growth in mice with HeLa tumor xenografts. In conclusion, our results indicated that fisetin exhibited anti-cancer effect and induced apoptosis in HeLa cell lines both in vitro and in vivo.

  13. Up-regulation of early growth response gene 1 (EGR-1) via ERK1/2 signals attenuates sulindac sulfide-mediated cytotoxicity in the human intestinal epithelial cells

    SciTech Connect

    Moon, Yuseok Yang, Hyun; Kim, Yung Bu

    2007-09-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and inflammation and have also received considerable attention because of their preventive effects against human cancer. However, the drug application is sometimes limited by the severe gastrointestinal ulcers and mucosal complications. In the present study, NSAID sulindac sulfide was investigated for the cytotoxic injury in the intestinal epithelial cells in association with an immediate inducible factor, early growth response gene 1 (EGR-1). Previously we reported that sulindac sulfide can suppress tumor cell invasion by inducing EGR-1. Extending the previous study, EGR-1 induction by sulindac sulfide was observed both in the non-transformed and transformed human intestinal epithelial cell lines. In terms of signaling pathway, ERK1/2 MAP kinases and its substrate Elk-1 transcription factor were involved in the sulindac sulfide-induced EGR-1 gene expression. Moreover, sulindac sulfide stimulated the nuclear translocation of the transcription factor EGR-1, which was also mediated by ERK1/2 signaling pathway. The roles of EGR-1 signals in the apoptotic cell death were assessed in the intestinal epithelial cells. Suppression of EGR-1 expression retarded cellular growth and colony forming activity in the intestinal epithelial cells. Moreover, induced EGR-1 ameliorated sulindac sulfide-mediated apoptotic cell death and enhanced the cellular survival. Taken all together, sulindac sulfide activated ERK1/2 MAP kinases which then mediated EGR-1 induction and nuclear translocation, all of which played important roles in the cellular survival from NSAID-mediated cytotoxicity in the human intestinal epithelial cells, implicating the protective roles of EGR-1 in the NSAID-mediated mucosal injuries.

  14. High insulin-induced down-regulation of Erk-1/IGF-1R/FGFR-1 signaling is required for oxidative stress-mediated apoptosis of adipose-derived stem cells.

    PubMed

    Scioli, Maria Giovanna; Cervelli, Valerio; Arcuri, Gaetano; Gentile, Pietro; Doldo, Elena; Bielli, Alessandra; Bonanno, Elena; Orlandi, Augusto

    2014-12-01

    Homeostasis of adipose tissue requires highly coordinated response between circulating factors and cell population. Human adult adipose-derived stem cells (ASCs) display multiple differentiation properties and are sensitive to insulin stimulation. Insulin resistance and high level of circulating insulin characterize patients with type 2 diabetes and obesity. At physiological concentration, insulin promoted proliferation and survival of ASCs in vitro, whereas high insulin level induced their dose-dependent proliferative arrest and apoptosis. Insulin-induced apoptotic commitment depended on the down-regulation of Erk-1, insulin growth factor-1 receptor (IGF-1R), and fibroblast growth factor receptor-1 (FGFR-1)-mediated signaling. Specific inhibition of Erk-1/2, IGF-1R, and FGFR activity promoted ASC apoptosis but did not increase insulin effects, whereas EGFR and ErbB2 inhibition potentiated insulin-induced apoptosis. FGFRs and EGFR inhibition reduced ASC adipogenic differentiation, whereas Erk-1/2 and IGF-1R inhibition was ineffective. Insulin-induced apoptosis associated to reactive oxygen species (ROS) accumulation and inhibition of NADPH oxidase 4 (Nox4) activity prevented ASC apoptosis. Moreover, specific inhibition of Erk-1/2, IGF-1R, and FGFR-1 activity promoted ROS generation and this effect was not cumulative with that of insulin alone. Our data indicate that insulin concentration is a critical regulatory switch between proliferation and survival of ASCs. High insulin level-induced apoptotic machinery involves Nox4-generated oxidative stress and the down-regulation of a complex receptor signaling, partially distinct from that influencing adipogenic differentiation of ASCs.

  15. Heterodimerization of apelin receptor and neurotensin receptor 1 induces phosphorylation of ERK(1/2) and cell proliferation via Gαq-mediated mechanism.

    PubMed

    Bai, Bo; Cai, Xin; Jiang, Yunlu; Karteris, Emmanouil; Chen, Jing

    2014-10-01

    Dimerization of G protein-coupled receptors (GPCRs) is crucial for receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin-converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C-terminal residues of vasoactive peptides including apelin-13, and neurotensin that activate the apelin receptor (APJ) and neurotensin receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co-immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK(1/2) activation and increased proliferation via activation of Gq α-subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK(1/2) activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.

  16. In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    SciTech Connect

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan

    2013-10-15

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{sub i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR

  17. Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

    SciTech Connect

    Ko, Jen-Chung; Tsai, Min-Shao; Weng, Shao-Hsing; Kuo, Ya-Hsun; Chiu, Yu-Fan; Lin, Yun-Wei

    2011-09-15

    Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: > Curcumin downregulates MKK-ERK-mediated Rad51 expression. > Curcumin enhances mitomycin C-induced cytotoxicity. > Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. > Rad51 inhibition enhances the chemosensitization of mitomycin C by

  18. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC-ERK1/2-mTOR signaling pathway.

    PubMed

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W

    2015-03-17

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

  19. HLA class I-mediated stress fiber formation requires ERK1/2 activation in the absence of an increase in intracellular Ca2+ in human aortic endothelial cells.

    PubMed

    Ziegler, Mary E; Jin, Yi-Ping; Young, Steven H; Rozengurt, Enrique; Reed, Elaine F

    2012-10-15

    Following transplantation, HLA class I antibodies targeting donor endothelium stimulate cell proliferation and migration, which contribute to the development of transplant vasculopathy and chronic allograft rejection. Dynamic remodeling of the actin cytoskeleton regulates cell proliferation and migration in endothelial cells (ECs), but the mechanism(s) involved remain incompletely understood. We explored anti-HLA class I antibody-mediated alterations of the cytoskeleton in human aortic ECs (HAECs) and contrasted these findings to thrombin-induced cytoskeleton remodeling. Our results identify two different signaling pathways leading to myosin light chain (MLC) phosphorylation in HAECs. Stimulation of HAECs with thrombin at 1 U/ml induced a robust elevation of intracellular Ca(2+) concentration, increased MLC phosphorylation, and promoted stress fiber formation via MLC kinase (MLCK) and Rho kinase (ROK) in an ERK-independent manner. In contrast, HAECs stimulated with HLA class I antibodies did not promote any detectable change in intracellular Ca(2+) concentration but instead induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. Stimulation of HAECs with low-dose thrombin (1 mU/ml) induced signaling cascades that were similar to stimulation with HLA class I antibodies. HLA class I antibodies also stimulated the translocation of mammalian target of rapamycin complex 2 (mTORC2) and ERK1/2 from the cytoplasm to the plasma membrane independently of stress fiber assembly. These findings identify novel roles for HLA class I signaling in ECs and provide new insights into the role of ERK1/2 and mTORC2 in cytoskeleton regulation, which may be important in promoting transplant vasculopathy, tumor angiogenesis, and atherosclerosis. PMID:22914643

  20. OM85-BV Induced the Productions of IL-1β, IL-6, and TNF-α via TLR4- and TLR2-Mediated ERK1/2/NF-κB Pathway in RAW264.7 Cells

    PubMed Central

    Luan, Hong; Zhang, Qian; Wang, Le; Wang, Chuanxiao; Zhang, Miao; Xu, Xiaoli; Zhou, Huan; Li, Xing'ai; Xu, Qing; He, Fan

    2014-01-01

    Broncho-Vaxom (OM85-BV) is an extract mixture from 8 strains of Gram+ and Gram− bacteria and plays an important role in anti-infection immune response by regulating macrophage activity and cytokine productions. However, the mechanism by which OM85-BV enhances the cytokine expression is still obscure. In this study, we evaluated the effects of OM85-BV on the productions of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in RAW264.7 murine macrophages. Exposure of RAW264.7 cells to 100 μg/mL OM85-BV upregulated the expression of IL-1β, IL-6, and TNF-α at the mRNA and protein levels in a time- and dose-dependent manner. In addition, OM85-BV induced extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor-kappa B (NF-κB) phosphorylation. Pretreatment with U0126 or Bay11-7082, respectively, could decrease IL-1β, IL-6, and TNF-α productions induced by OM85-BV. Application of Toll-like receptor (TLR) 4 or TLR2 small-interfering RNA (siRNA) into RAW264.7 cells could inhibit the productions of cytokines and ERK1/2 and NF-κB phosphorylation induced by OM85-BV. Consistent with this, downregulating either myeloid differentiation factor 88 (MyD88) or TRIF-related adaptor molecule (TRAM) gene with MyD88-siRNA or TRAM-siRNA separately could reduce the productions of cytokines and ERK1/2 and NF-κB phosphorylation induced by OM85-BV. Our study demonstrated that the productions of IL-1β, IL-6, and TNF-α induced by OM85-BV in RAW264.7 cells were through TLR4 and TLR2 signaling pathway-mediated activation of ERK1/2 and NF-κB. PMID:24605772

  1. Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

    PubMed Central

    Zhong, Weixia; Hutchinson, Tarun E.; Chebolu, Seetha; Darmani, Nissar A.

    2014-01-01

    that Ca2+ mobilization via extracellular Ca2+ influx through 5-HT3Rs/L-type Ca2+ channels, and intracellular Ca2+ release via RyRs on ER, initiate Ca2+-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis. PMID:25121483

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

    PubMed

    Malik, Shaily; Saha, Rinki; Seth, Pankaj

    2014-06-01

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

  3. Whole body vibration improves osseointegration by up-regulating osteoblastic activity but down-regulating osteoblast-mediated osteoclastogenesis via ERK1/2 pathway.

    PubMed

    Zhou, Yi; Guan, Xiaoxu; Liu, Tie; Wang, Xinhua; Yu, Mengfei; Yang, Guoli; Wang, Huiming

    2015-02-01

    Due to the reduction in bone mass and deterioration in bone microarchitecture, osteoporosis is an important risk factor for impairing implant osseointegration. Recently, low-magnitude, high-frequency (LMHF) vibration (LM: <1×g; HF: 20-90Hz) has been shown to exhibit anabolic, but anti-resorptive effects on skeletal homeostasis. Therefore, we hypothesized that LMHF loading, in terms of whole body vibration (WBV), may improve implant fixation under osteoporotic status. In the in vivo study, WBV treatment (magnitude: 0.3g, frequency: 40Hz, time: 30min/12h, 5days/week) was applied after hydroxyapatite-coated titanium implants were inserted in the bilateral tibiae of ovariectomized rats. The bone mass and the osteospecific gene expressions were measured at 12weeks post implantation. In the in vitro study, the cellular and molecular mechanisms underlying osteoblastic and osteoclastic activities were fully investigated using various experimental assays. Micro-CT examination showed that WBV could enhance osseointegration by improving microstructure parameters surrounding implants. WBV-regulated gene levels in favor of bone formation over resorption may be the reason for the favorable adaptive bone remolding on bone-implant surface. The in vitro study showed that vibration (magnitude: 0.3g, frequency: 40Hz, time: 30min/12h) up-regulated osteoblast differentiation, matrix synthesis and mineralization. However, mechanically regulated osteoclastic activity was mainly through the effect on osteoblastic cells producing osteoclastogenesis-associated key soluble factors, including RANKL and M-CSF. Osteoblasts were therefore the direct target cells during the mechanotransduction process. The ERK1/2 pathway was demonstrated to play an essential role in vibration-induced enhancement of bone formation and decreased bone resorption. Our data suggests that WBV was a helpful non-pharmacological intervention for improving osseointegration under osteoporosis.

  4. The Dictyostelium MAPK ERK1 is phosphorylated in a secondary response to early developmental signaling

    PubMed Central

    Schwebs, David J.; Hadwiger, Jeffrey A.

    2014-01-01

    Previous reports have suggested that the two mitogen-activated protein kinases (MAPKs) in Dictyostelium discoideum, ERK1 and ERK2, can be directly activated in response to external cAMP even though these MAPKs play different roles in the developmental life cycle. To better characterize MAPK regulation, the levels of phosphorylated MAPKs were analyzed in response to external signals. Only ERK2 was rapidly phosphorylated in response to the chemoattractants, cAMP and folate. In contrast, the phosphorylation of ERK1 occurred as a secondary or indirect response to these stimuli and this phosphorylation was enhanced by cell-cell interactions, suggesting that other external signals can activate ERK1. The phosphorylation of ERK1 or ERK2 did not require the function of the other MAPK in these responses. Folate stimulation of a chimeric population of erk1− and gα4− cells revealed that the phosphorylation of ERK1 could be mediated through an intercellular signal other than folate. Loss of ERK1 function suppressed the developmental delay and the deficiency in anterior cell localization associated with gα5− mutants suggesting that ERK1 function can be down regulated through Gα5 subunit-mediated signaling. However, no major changes in the phosphorylation of ERK1 were observed in gα5− cells suggesting that the Gα5 subunit signaling pathway does not regulate the phosphorylation of ERK1. These findings suggest that the activation of ERK1 occurs as a secondary response to chemoattractants and that other cell-cell signaling mechanisms contribute to this activation. Gα5 subunit signaling can down regulate ERK1 function to promote prestalk cell development but not through major changes to the level of phosphorylated ERK1. PMID:25451080

  5. MAPK Signaling and ERK1/2 bistability in Asthma

    PubMed Central

    Alam, Rafeul; Gorska, Magdalena M.

    2010-01-01

    Mitogen-activated protein kinases (MAPK) integrate signals from numerous receptors and translate these signals into cell functions. MAPKs are critical for immune cell metabolism, migration, production of pro-inflammatory mediators, survival, and differentiation. We provide a concise review of the involvement of MAPK in important cells of the immune system. Certain cell functions e.g. production of pro-inflammatory mediators resolve quickly and may require a transient MAPK activation, other processes such as cell differentiation and long-term survival may require persistent MAPK signal. The persistent MAPK signal is frequently a consequence of positive feedback loops or double negative feedback loops which perpetuate the signal after removal of an external cell stimulus. This self-perpetuated activation of a signaling circuit is a manifestation of its bistability. Bistable systems can exist in “on” and “off” states and both states are stable. We have demonstrated the existence of self-perpetuated activation mechanism for ERK1/2 in bronchial epithelial cells. This sustained activation of ERK1/2 supports long-term survival of these cells and primes them for cytokine transcription. ERK1/2 bistability arises from repetitive stimulation of the cell. The repeated stimulation (e.g. repeated viral infection or repeated allergen exposure) seems to be a common theme in asthma and other chronic illnesses. We thus hypothesize that the self-perpetuated ERK1/2 signal plays an important role in the pathogenesis of asthma. PMID:21121982

  6. Specifically targeting ERK1 or ERK2 kills Melanoma cells

    PubMed Central

    2012-01-01

    Background Overcoming the notorious apoptotic resistance of melanoma cells remains a therapeutic challenge given dismal survival of patients with metastatic melanoma. However, recent clinical trials using a BRAF inhibitor revealed encouraging results for patients with advanced BRAF mutant bearing melanoma, but drug resistance accompanied by recovery of phospho-ERK (pERK) activity present challenges for this approach. While ERK1 and ERK2 are similar in amino acid composition and are frequently not distinguished in clinical reports, the possibility they regulate distinct biological functions in melanoma is largely unexplored. Methods Rather than indirectly inhibiting pERK by targeting upstream kinases such as BRAF or MEK, we directly (and near completely) reduced ERK1 and ERK2 using short hairpin RNAs (shRNAs) to achieve sustained inhibition of pERK1 and/or pERK2. Results and discussion Using A375 melanoma cells containing activating BRAFV600E mutation, silencing ERK1 or ERK2 revealed some differences in their biological roles, but also shared roles by reduced cell proliferation, colony formation in soft agar and induced apoptosis. By contrast, chemical mediated inhibition of mutant BRAF (PLX4032) or MEK (PD0325901) triggered less killing of melanoma cells, although they did inhibit proliferation. Death of melanoma cells by silencing ERK1 and/or ERK2 was caspase dependent and accompanied by increased levels of Bak, Bad and Bim, with reduction in p-Bad and detection of activated Bax levels and loss of mitochondrial membrane permeability. Rare treatment resistant clones accompanied silencing of either ERK1 and/or ERK2. Unexpectedly, directly targeting ERK levels also led to reduction in upstream levels of BRAF, CRAF and pMEK, thereby reinforcing the importance of silencing ERK as regards killing and bypassing drug resistance. Conclusions Selectively knocking down ERK1 and/or ERK2 killed A375 melanoma cells and also increased the ability of PLX4032 to kill A375 cells

  7. The naturally occurring biflavonoid, ochnaflavone, inhibits LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-kappaB regulation, in RAW264.7 cells.

    PubMed

    Suh, Seok-Jong; Chung, Tae-Wook; Son, Min-Jung; Kim, Sung-Hoon; Moon, Tae Chul; Son, Kun Ho; Kim, Hyun Pyo; Chang, Hyeun Wook; Kim, Cheorl-Ho

    2006-03-15

    Ochnaflavone (OC), a naturally occurring biflavonoid with anti-inflammatory activity [S.J. Lee, J.H. Choi, H.W. Chang, S.S. Kang, H.P. Kim. Life Sci. 57(6), 1995, 551-558], was isolated from Lonicera japonica and its effects on inducible nitric oxide synthase (iNOS) gene expression was examined in RAW264.7 cells. U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly down-regulated lipopolysaccharide (LPS)-induced iNOS expression and promoter activity. Transactivation of LPS-stimulated NF-kappaB was inhibited by U0126. These results suggest that the transcription factor NF-kappaB is involved in ERK-mediated iNOS regulation and that activation of the Ras/ERK pathway contributes to the induction of iNOS expression in RAW264.7 cells in response to LPS. OC treatment inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the LPS-induced expression of iNOS. These inhibitory effects were associated with reduced ERK1/2 activity. OC inhibited the phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase. The findings herein show that the inhibition of LPS-induced ERK1/2 activation may be a contributing factor to the main mechanisms by which OC inhibits RAW264.7. To clarify the mechanistic basis for its ability to inhibit iNOS induction, we examined the effect of OC on the transactivation of the iNOS gene by luciferase reporter activity using the -1588 flanking region. OC potently suppressed reporter gene activity. We also report here, for the first time, that LPS-induced iNOS expression was abolished by OC in RAW264.7 cells through by blocking the inhibition of transcription factor NF-kappaB binding activities. These activities are associated with the down-regulation of inhibitor kappaB (IkappaB) kinase (IKK) activity by OC (6 microM), thus inhibiting LPS-induced phosphorylation as well as the degradation of IkappaBalpha. These findings suggest that the inhibition of LPS

  8. CIH-induced neurocognitive impairments are associated with hippocampal Ca(2+) overload, apoptosis, and dephosphorylation of ERK1/2 and CREB that are mediated by overactivation of NMDARs.

    PubMed

    Wang, Jing; Ming, Hong; Chen, Rui; Ju, Jing-Mei; Peng, Wan-da; Zhang, Guo-Xing; Liu, Chun-Feng

    2015-11-01

    Chronic intermittent hypoxia (CIH) is commonly seen in patients with obstructive sleep apnea, and has been hypothesized to underlie the neurocognitive dysfunction in these patients. However, its cellular and molecular mechanisms remain to be defined. The present study aimed to investigate, in a mouse CIH model, the role of NMDA receptor (NMDAR) activation in mediating the CIH-induced neurocognitive impairments, caspase expression and dysregulated Ca(2+) signaling pathways in hippocampus. Male ICR mice (n=45) were exposed to CIH (8h/day) or room air (control) for 4 weeks. After 4-week treatment, neurobehavioral assessments were performed by Morris water maze test, hippocampal [Ca(2+)]i was evaluated by flow cytometry; and protein expressions of caspase-3, caspase-9, PARP, p-ERK1/2 and p-CREB in hippocampus were measured by Western blotting. Our results showed that, compared to control animals, 4-week exposure to CIH produced significant spatial learning and memory impairments in CIH mice. Increased caspase expression in hippocampus was observed in CIH mice associated with significant elevation of [Ca(2+)]i and dephosphorylation of ERK and CREB expression. When the NMDAR antagonist memantine was administered by intraperitoneal injection prior to daily exposure to CIH, at a sub-therapeutic dose of 5mg/kg/day not shown to impact the neurobehavioral performance in control animals, the neurocognitive impairments as well as the neurobiochemical changes were abolished or normalized in the CIH mice. Our study suggests that overactivation of NMDARs and the Ca(2+) overload-dependent ERK/CREB dysregulation is one of the important mechanisms in mediating the CIH-induced neurocognitive impairments.

  9. Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells.

    PubMed

    Jeon, Bo Kyung; Kwon, Kihwan; Kang, Jihee Lee; Choi, Youn-Hee

    2015-08-03

    Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress.

  10. Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2.

    PubMed

    Marchbank, Tania; Mahmood, Asif; Playford, Raymond J

    2013-08-01

    Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

  11. ERK1/2 regulate the balance between eccentric and concentric cardiac growth

    PubMed Central

    Kehat, Izhak; Davis, Jennifer; Tiburcy, Malte; Accornero, Federica; Saba-El-Leil, Marc K.; Maillet, Marjorie; York, Allen J.; Lorenz, John N.; Zimmermann, Wolfram H.; Meloche, Sylvain; Molkentin, Jeffery D.

    2011-01-01

    /2 signaling cascade in regulating the cardiac hypertrophic response. While activation of the ERK pathway induced concentric hypertrophy, inhibition of this pathway resulted in eccentric hypertrophy. Using cardiomyocytes isolated from these mouse models, and using ex vivo culture models we show that these effects were mediated directly by ERK signaling. Greater ERK1/2 signaling directly programmed myocyte thickening while inhibition of ERK1/2 signaling promoted myocyte lengthening. Thus, this study sheds light on the molecular mechanisms that are partially responsible for the differential hypertrophic response of the heart to an increase in preload versus afterload. PMID:21127295

  12. Activation of PKC{beta}{sub II} and PKC{theta} is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

    SciTech Connect

    Heo, Kyung-Sun; Kim, Dong-Uk; Kim, Lila; Nam, Miyoung; Baek, Seung-Tae; Park, Song-Kyu; Park, Youngwoo; Myung, Chang-Seon; Hwang, Sung-Ook Hoe, Kwang-Lae

    2008-03-28

    Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKC{beta}{sub II} and PKC{theta} from cytosol to plasma membrane, and inhibition of PKC{beta}{sub II} and PKC{theta} decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKC{beta}{sub II} and PKC{theta}, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKC{beta}{sub II} and PKC{theta}. Inhibition of PKC{beta}{sub II} or PKC{theta}, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKC{theta} in VSMC proliferation is unique.

  13. EPO gene expression induces the proliferation, migration and invasion of bladder cancer cells through the p21WAF1‑mediated ERK1/2/NF-κB/MMP-9 pathway.

    PubMed

    Park, Sung Lyea; Won, Se Yeon; Song, Jun-Hui; Kim, Wun-Jae; Moon, Sung-Kwon

    2014-11-01

    Erythropoietin (EPO) is a cytokine that modulates the production of red blood cells. Previous studies have contradicted the assumed role of EPO in tumor cell proliferation. In the present study, we investigated the effect of EPO in the proliferation, migration and invasion that is involved in the signaling pathways and cell-cycle regulation of bladder cancer 5637 cells. The results showed that an overexpression of the EPO gene has a potent stimulatory effect on DNA synthesis, migration and invasion. EPO gene expression increased the expression of matrix metalloproteinase (MMP)-9 via the binding activity of NF-κB, AP-1 and Sp-1 in 5637 cells. The transfection of 5637 cells with the EPO gene induced the phosphorylation of ERK1/2. Treatment with ERK1/2 inhibitor U0126 significantly inhibited the increased proliferation, migration and invasion of EPO gene-transfected cells. U0126 treatment suppressed the induction of MMP-9 expression through NF-κB binding activity in EPO gene transfectants. In addition, EPO gene expression was correlated with the upregulation of cyclins/CDKs and the upregulation of the CDK inhibitor p21WAF1 expression. Finally, the inhibition of p21WAF1 function by siRNA blocked the proliferation, migration, invasion and phosphorylation of ERK1/2 signaling, as well as MMP-9 expression and activation of NF-κB in EPO gene-transfected cells. These novel findings suggest that the molecular mechanisms of EPO contribute to the progression and development of bladder tumors. PMID:25175278

  14. Melittin inhibits TGF-β-induced pro-fibrotic gene expression through the suppression of the TGFβRII-Smad, ERK1/2 and JNK-mediated signaling pathway.

    PubMed

    Park, Su-Hyun; Cho, Hyun-Ji; Jeong, Yun-Jeong; Shin, Jae-Moon; Kang, Jeong-Han; Park, Kwan-Kyu; Choe, Jung-Yoon; Park, Yoon-Yub; Bae, Young-Seuk; Han, Sang-Mi; Moon, Sung-Kwon; Kim, Wun-Jae; Choi, Yung Hyun; Chang, Young-Chae

    2014-01-01

    Renal fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins such as type I collagen, fibronectin, and by the increased expression of PAI-1. This study evaluated the anti-fibrotic effect of bee venom and its major compounds (melittin and apamin) on TGF-β-induced pro-fibrotic gene expression. Bee venom and melittin significantly suppressed type I collagen, fibronectin, and PAI-1 protein expression in the TGF-β-treated kidney fibroblast. However, apamin only inhibited the expression of fibronectin and type I collagen. These results indicated that the inhibitory effects of bee venom on TGF-β-induced pro-fibrotic gene expression are caused by melittin. Moreover, we attempted to elucidate mechanisms underlying the anti-fibrotic effect of melittin. Melittin dramatically inhibited the phosphorylation of TGFβRII and Smad2/3. Also, melittin inhibited the phosphorylation of ERK1/2 and JNK, but not the phosphorylation of PI3K, Akt, and p38. These results suggested that melittin inhibits TGF-β-induced pro-fibrotic genes expression through the suppression of TGFβR-Smad2/3, ERK1/2, and JNK phosphorylation, and melittin can be used as a clinical drug for the treatment of fibrosis associated with renal diseases.

  15. Growth-stimulatory activity of TIMP-2 is mediated through c-Src activation followed by activation of FAK, PI3-kinase/AKT, and ERK1/2 independent of MMP inhibition in lung adenocarcinoma cells.

    PubMed

    Kim, Han Ie; Lee, Hyun-Sung; Kim, Tae Hyun; Lee, Ju-Seog; Lee, Seung-Taek; Lee, Seo-Jin

    2015-12-15

    Tissue inhibitors of metalloproteinases (TIMPs) control extracellular matrix (ECM) homeostasis by inhibiting the activity of matrix metalloproteinases (MMPs), which are associated with ECM turnover. Recent studies have revealed that TIMPs are implicated in tumorigenesis in both MMP-dependent and MMP-independent manners. We examined a mechanism by which TIMP-2 stimulated lung adenocarcinoma cell proliferation, independent of MMP inhibition. The stimulation of growth by TIMP-2 in A549 cells required c-Src kinase activation. c-Src kinase activity, induced by TIMP-2, concomitantly increased FAK, phosphoinositide 3-kinase (PI3-kinase)/AKT, and ERK1/2 activation. Selective knockdown of integrin α3β1, known as a TIMP-2 receptor, did not significantly change TIMP-2 growth promoting activity. Furthermore, we showed that high TIMP-2 expression in lung adenocarcinomas is associated with a worse prognosis from multiple cohorts, especially for stage I lung adenocarcinoma. Through integrated analysis of The Cancer Genome Atlas data, TIMP-2 expression was significantly associated with the alteration of driving genes, c-Src activation, and PI3-kinase/AKT pathway activation. Taken together, our results demonstrate that TIMP-2 stimulates lung adenocarcinoma cell proliferation through c-Src, FAK, PI3-kinase/AKT, and ERK1/2 pathway activation in an MMP-independent manner.

  16. Distinct Pathways of ERK1/2 Activation by Hydroxy-Carboxylic Acid Receptor-1

    PubMed Central

    Li, Guo; Wang, Hui-qian; Wang, Li-hui; Chen, Ru-ping; Liu, Jun-ping

    2014-01-01

    Mechanistic investigations have shown that, upon agonist activation, hydroxy-carboxylic acid receptor-1(HCA1) couples to a Gi protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for HCA1 signaling remain largely unknown. Using CHO-K1 cells stably expressing HCA1, and L6 cells, which endogenously express rat HCA1 receptors, we found that activation of ERK1/2 by HCA1 was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that HCA1 induced ERK1/2 activation via the extracellular Ca2+, PKC and IGF-I receptor transactivation-dependent pathways. In addition, we observed that pretreated the cells with M119K, an inhibitor of Gβγ subunit-dependent signaling, effectively attenuated the ERK1/2 activation triggered by HCA1, suggesting a critical role for βγ-subunits in HCA1-activated ERK1/2 phosphorylation. Furthermore, the present results also indicated that the arrestin2/3 were not required for ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to agonist, HCA1 receptors initially activate Gi, leading to dissociation of the Gβγ subunit from activated Gi, and subsequently induce ERK1/2 activation via two distinct pathways: one PKC-dependent pathway and the other IGF-IR transactivation-dependent pathway. Our results provide the first in-depth evidence that defines the molecular mechanism of HCA1-mediated ERK1/2 activation. PMID:24671202

  17. The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus.

    PubMed

    Chen, Wei-Hong; Zeng, Rong; Lo, Wen-Cheng; Tina Chen, Szu-Yu; Lai, Tung-Yuan; Williams, David F; Deng, Win-Ping

    2012-11-01

    Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10(-8)-10(-6) M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations.

  18. How ERK1/2 Activation Controls Cell Proliferation and Cell Death Is Subcellular Localization the Answer?

    PubMed Central

    Mebratu, Yohannes; Tesfaigzi, Yohannes

    2009-01-01

    Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase super family that can mediate cell proliferation and apoptosis. The Ras–Raf–MEK–ERK signaling cascade controlling cell proliferation has been well studied but the mechanisms involved in ERK1/2-mediated cell death are largely unknown. This review focuses on recent papers that define ERK1/2 translocation to the nucleus and the proteins involved in the cytosolic retention of activated ERK1/2. Cytosolic retention of ERK1/2 denies access to the transcription factor substrates that are responsible for the mitogenic response. In addition, cytosolic ERK1/2, besides inhibiting survival and proliferative signals in the nucleus, potentiates the catalytic activity of some proapoptotic proteins such as DAP kinase in the cytoplasm. Studies that further define the function of cytosolic ERK1/2 and its cytosolic substrates that enhance cell death will be essential to harness this pathway for developing effective treatments for cancer and chronic inflammatory diseases. PMID:19282669

  19. Altered ERK1/2 Signaling in the Brain of Learned Helpless Rats: Relevance in Vulnerability to Developing Stress-Induced Depression

    PubMed Central

    Dwivedi, Yogesh; Zhang, Hui

    2016-01-01

    Extracellular signal-regulated kinase 1/2- (ERK1/2-) mediated cellular signaling plays a major role in synaptic and structural plasticity. Although ERK1/2 signaling has been shown to be involved in stress and depression, whether vulnerability to develop depression is associated with abnormalities in ERK1/2 signaling is not clearly known. The present study examined ERK1/2 signaling in frontal cortex and hippocampus of rats that showed vulnerability (learned helplessness, (LH)) or resiliency (non-learned helplessness, (non-LH)) to developing stress-induced depression. In frontal cortex and hippocampus of LH rats, we found that mRNA and protein expressions of ERK1 and ERK2 were significantly reduced, which was associated with their reduced activation and phosphorylation in cytosolic and nuclear fractions, where ERK1 and ERK2 target their substrates. In addition, ERK1/2-mediated catalytic activities and phosphorylation of downstream substrates RSK1 (cytosolic and nuclear) and MSK1 (nuclear) were also lower in the frontal cortex and hippocampus of LH rats without any change in their mRNA or protein expression. None of these changes were evident in non-LH rats. Our study indicates that ERK1/2 signaling is differentially regulated in LH and non-LH rats and suggests that abnormalities in ERK1/2 signaling may be crucial in the vulnerability to developing depression. PMID:26839717

  20. ERK2 Alone Drives Inflammatory Pain But Cooperates with ERK1 in Sensory Neuron Survival

    PubMed Central

    O'Brien, Daniel E.; Alter, Benedict J.; Satomoto, Maiko; Morgan, Clinton D.; Davidson, Steve; Vogt, Sherri K.; Norman, Megan E.; Gereau, Graydon B.; Demaro, Joseph A.; Landreth, Gary E.; Golden, Judith P.

    2015-01-01

    Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are highly homologous yet distinct components of signal transduction pathways known to regulate cell survival and function. Recent evidence indicates an isoform-specific role for ERK2 in pain processing and peripheral sensitization. However, the function of ERK2 in primary sensory neurons has not been directly tested. To dissect the isoform-specific function of ERK2 in sensory neurons, we used mice with Cre-loxP-mediated deletion of ERK2 in Nav1.8+ sensory neurons that are predominantly nociceptors. We find that ERK2, unlike ERK1, is required for peripheral sensitization and cold sensation. We also demonstrate that ERK2, but not ERK1, is required to preserve epidermal innervation in a subset of peptidergic neurons. Additionally, deletion of both ERK isoforms in Nav1.8+ sensory neurons leads to neuron loss not observed with deletion of either isoform alone, demonstrating functional redundancy in the maintenance of sensory neuron survival. Thus, ERK1 and ERK2 exhibit both functionally distinct and redundant roles in sensory neurons. SIGNIFICANCE STATEMENT ERK1/2 signaling affects sensory neuron function and survival. However, it was not clear whether ERK isoform-specific roles exist in these processes postnatally. Previous work from our laboratory suggested either functional redundancy of ERK isoforms or a predominant role for ERK2 in pain; however, the tools to discriminate between these possibilities were not available at the time. In the present study, we use new genetic knock-out lines to demonstrate that ERK2 in sensory neurons is necessary for development of inflammatory pain and for postnatal maintenance of peptidergic epidermal innervation. Interestingly, postnatal loss of both ERK isoforms leads to a profound loss of sensory neurons. Therefore, ERK1 and ERK2 display both functionally distinct and redundant roles in sensory neurons. PMID:26109671

  1. pERK 1/2 inhibit Caspase-8 induced apoptosis in cancer cells by phosphorylating it in a cell cycle specific manner.

    PubMed

    Mandal, Ranadip; Raab, Monika; Matthess, Yves; Becker, Sven; Knecht, Rainald; Strebhardt, Klaus

    2014-03-01

    ERK 1/2 are found to be hyperactive in many cancers. Active ERK 1/2 (pERK 1/2) are known to protect cancer cells from undergoing death receptor-mediated apoptosis, although the mechanism(s) behind this is poorly understood. Through in vitro kinase assays and mass-spectrometry we demonstrate that pERK 1/2 can phosphorylate pro-Caspase-8 at S387. Also, in EGFR-overexpressing Type I and II ovarian and breast cancer cell lines respectively, ERK 1/2 remain active only during the interphase. During this period, pERK 1/2 could inhibit Trail-induced apoptosis, most effectively during the G1/S phase. By knocking-down the endogenous pro-Caspase-8 using RNAi and replacing it with its non-phosphorylatable counterpart (S387A), a significant increase in Caspase-8 activity upon Trail stimulation was observed, even in the presence of pERK 1/2. Taken together, we propose that a combination of Trail and an inhibitor of ERK 1/2 activities could potentially enhance of Trail's effectiveness as an anti-cancer agent in ERK 1/2 hyperactive cancer cells.

  2. The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

    PubMed

    Perez-Aso, M; Segura, V; Montó, F; Barettino, D; Noguera, M A; Milligan, G; D'Ocon, P

    2013-10-01

    We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.

  3. Differential expression profiles and roles of inducible DUSPs and ERK1/2-specific constitutive DUSP6 and DUSP7 in microglia.

    PubMed

    Ham, Ji-Eun; Oh, Eun-Kyung; Kim, Dong-Hoon; Choi, Sang-Hyun

    2015-11-13

    Dual-specificity phosphatases (DUSPs) show distinct substrate preferences for specific MAPKs. DUSPs sharing a substrate preference for ERK1/2 may be classified as inducible or constitutive. In contrast to the inducible DUSPs which also dephosphorylate p38 MAPK and JNK in the major inflammatory pathways, constitutive DUSP6 and DUSP7 are specific to ERK1/2 and have not been studied in microglia and other immune cells to date. In the present study, we differentiated mRNA expression profiles of inducible and constitutive DUSPs that dephosphorylate ERK1/2 in microglia. Lipopolysaccharide (LPS) at 1 ng/ml induced prompt phosphorylation of ERK1/2 with peak induction at 30 min. LPS induced expression of DUSP1, DUSP2, and DUSP5 within 60 min, whereas DUSP4 expression was induced more slowly. DUSP6 and DUSP7 exhibited constitutive basal expression, which decreased immediately after LPS stimulation but subsequently returned to basal levels. The expression of DUSP6 and DUSP7 was regulated inverse to the phosphorylation of ERK1/2 in LPS-stimulated microglia. Therefore, we next investigated the correlation between DUSP6 and DUSP7 expression and ERK1/2 phosphorylation in resting and LPS-stimulated microglia. Inhibition of the ERK1/2 pathway by PD98059 and FR180204 resulted in a decrease in DUSP6 and DUSP7 expression, both in resting and LPS-stimulated microglia. These inhibitors partially blocked the LPS-induced expression of DUSP1, DUSP2, and DUSP4, but had no effect on DUSP5. Finally, we examined the role of DUSP6 activity in the downregulation of ERK1/2 phosphorylation. BCI, an inhibitor of DUSP6, increased the phosphorylation of ERK1/2. However, pretreatment with BCI inhibited the LPS-induced phosphorylation of ERK1/2. These results demonstrate that constitutive DUPS6 and DUSP7 expression was downregulated inverse to the expression of inducible DUSPs and the phosphorylation of ERK1/2 in LPS-stimulated microglia. The expression of DUPS6 and DUSP7 was mediated by ERK1

  4. Analyzing ERK 1/2 signalling and targets.

    PubMed

    Brietz, Alexandra; Schuch, Kristin Verena; Wangorsch, Gaby; Lorenz, Kristina; Dandekar, Thomas

    2016-07-19

    The ERK cascade (e.g. Raf-1) protects the heart from cell death and ischemic injury but can also turn maladaptive. Furthermore, an additional autophosphorylation of ERK2 at Thr188 (Erk1 at Thr208) allows ERK to phosphorylate nuclear targets involved in hypertrophy, stressing this additional phosphorylation as a promising pharmacological target. An in silico model was assembled and setup to reproduce different phosphorylation states of ERK 1/2 and various types of stimuli (hypertrophic versus non-hypertrophic). Synergistic and antagonistic receptor stimuli can be predicted in a semi-quantitative model, simulated time courses were experimentally validated. Furthermore, we detected new targets of ERK 1/2, which possibly contribute to the development of pathological hypertrophy. In addition we modeled further interaction partners involved in the protective and maladaptive cascade. Experimental validation included different gene expression data sets supporting key components and novel interaction partners as well as time courses in chronic heart failure.

  5. Loading-related regulation of transcription factor EGR2/Krox-20 in bone cells is ERK1/2 protein-mediated and prostaglandin, Wnt signaling pathway-, and insulin-like growth factor-I axis-dependent.

    PubMed

    Zaman, Gul; Sunters, Andrew; Galea, Gabriel L; Javaheri, Behzad; Saxon, Leanne K; Moustafa, Alaa; Armstrong, Victoria J; Price, Joanna S; Lanyon, Lance E

    2012-02-01

    Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression. In UMR106 cells, inhibition of β1/β3 integrin function had no effect on strain-related Egr2 expression, but it was inhibited by a COX2-selective antagonist and imitated by exogenous prostaglandin E2 (PGE2). This response to PGE(2) was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signaling, or LiCl had any effect on Egr2 mRNA expression, but it was increased by both insulin-like growth factor-1 and high, but not low, dose parathyroid hormone and exogenous Wnt-3a. The increases by strain, PGE2, Wnt-3a, and phorbol 12-myristate 13-acetate were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signaling pathways that constitute early responses of bone cells to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent "instructions" for adaptive (re)modeling is likely to depend upon their contextual activation, suppression, and interaction probably on more than one occasion. PMID:22049075

  6. Redox-regulation of Erk1/2-directed phosphatase by reactive oxygen species: role in signaling TPA-induced growth arrest in ML-1 cells.

    PubMed

    Traore, Kassim; Sharma, Rajni; Thimmulappa, Rajesh K; Watson, Walter H; Biswal, Shyam; Trush, Michael A

    2008-07-01

    Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.

  7. Role of ERK1/2 activation on itch sensation induced by bradykinin B1 activation in inflamed skin

    PubMed Central

    Chen, Yuanzhen; Jiang, Shuyan; Liu, Yuying; Xiong, Jialing; Liang, Jiexian; Ji, Wenjin

    2016-01-01

    It has previously been demonstrated that bradykinin receptor B1 (B1R) agonists evoke an itch-related scratching response in inflamed skin via the B1 receptor; however, the mechanisms responsible for this abnormal itch sensation remain unclear. Therefore, the present study utilized a complete Freund's adjuvant (CFA)-induced mouse model of inflammation to elucidate the mechanisms responsible. Over a period of 30 min, scratching behavior was quantified by the number of hind limb scratches of the area surrounding the drug injection site on the neck. Furthermore, western blot analysis was used to investigate the potential role of extracellular signal-regulated kinase (ERK) 1/2 signaling as a mediator of itch in CFA-treated mice. The results demonstrated that CFA-induced inflammation at the back of the neck is associated with sustained enhancement of ERK1/2 activation in the spinal cord. Moreover, B1R agonist treatment resulted in increased expression of phosphorylated ERK1/2 in the spinal cord, which peaked at 45 min. Consistent with these findings, inhibition of either mitogen-activated protein/ERK kinase or ERK1/2, as well as inhibition of ERK1/2 activation following inflammation, attenuated B1 receptor-mediated scratching responses to a greater extent, as compared with control mice. Collectively, the results of the present study indicated that enhanced and persistent ERK1/2 activation in the spinal cord may be required to induce a scratching response to B1R agonists following CFA-induced inflammation. PMID:27446253

  8. Effects of environmental enrichment on ERK1/2 phosphorylation in the rat prefrontal cortex following nicotine-induced sensitization or nicotine self-administration.

    PubMed

    Gomez, Adrian M; Sun, Wei-Lun; Midde, Narasimha M; Harrod, Steven B; Zhu, Jun

    2015-01-01

    Rats raised in an enriched condition (EC) exhibit alterations in the neurobiological and behavioral response to nicotine compared with rats reared in an impoverished condition (IC) or a standard condition (SC). The current study determined whether environmental enrichment differentially regulates extracellular signal-regulated kinase1/2 (ERK1/2) activity in the prefrontal cortex in rats following nicotine sensitization or nicotine self-administration. Under the saline control condition, EC rats displayed diminished baseline activity and greater sensitization to repeated administration of nicotine compared with IC and SC rats. After repeated saline injections, the basal levels of phosphorylated ERK1/2 (pERK1/2) were higher in EC compared with IC and SC rats, which was negatively correlated with their respective baseline activities. Repeated nicotine (0.35 mg/kg) injections induced pERK1/2 to similar levels in SC and IC rats; however, the induction of pERK1/2 in EC rats by nicotine was not significantly different from saline controls, owing to their high baseline. In the self-administration paradigm, EC rats self-administered less nicotine (0.03 mg/kg/infusion) relative to IC or SC rats on a fixed ratio-1 schedule of reinforcement. Accordingly, no differences in pERK1/2 were found between EC and IC rats self-administering saline, whereas nicotine self-administration resulted in an increase in pERK1/2 in IC rats but not in EC rats. Furthermore, the levels of pERK1/2 in EC and IC rats were positively correlated with their respective total number of nicotine infusions. Thus, these findings suggest that environmental enrichment alters the basal and nicotine-mediated pERK1/2, which may contribute to enrichment-induced behavioral alterations in response to nicotine.

  9. Effects of environmental enrichment on ERK1/2 phosphorylation in the rat prefrontal cortex following nicotine-induced sensitization or nicotine self-administration

    PubMed Central

    Gomez, Adrian M.; Sun, Wei-Lun; Midde, Narasimha M.; Harrod, Steven B.; Zhu, Jun

    2014-01-01

    Rats raised in an enriched condition (EC) exhibit alterations in the neurobiological and behavioral response to nicotine compared to rats reared in an impoverished condition (IC) or a standard condition (SC). The current study determined whether environmental enrichment differentially regulates extracellular signal-regulated kinase1/2 (ERK1/2) activity in the prefrontal cortex (PFC) in rats following nicotine sensitization or nicotine self-administration. Under the saline control condition, EC rats displayed diminished baseline activity, and greater sensitization to repeated administration of nicotine compared to IC and SC rats. After repeated saline injections, the basal levels of phosphorylated ERK1/2 (pERK1/2) were higher in EC compared to IC and SC rats, which was negatively correlated with their respective baseline activities. Repeated nicotine (0.35 mg/kg) injections induced pERK1/2 to similar levels in SC and IC rats; however, the induction of pERK1/2 in EC rats by nicotine was not significantly different from saline controls, owing to their high baseline. In the self-administration paradigm, EC rats self-administered less nicotine (0.03 mg/kg/infusion) relative to IC or SC rats on a fixed ratio-1 schedule of reinforcement. Accordingly, no differences in pERK1/2 were found between EC and IC rats self-administering saline, whereas nicotine self-administration resulted in an increase in pERK1/2 in IC rats but not in EC rats. Furthermore, the levels of pERK1/2 in EC and IC rats were positively correlated with their respective total number of nicotine infusions. Thus, these findings suggest that environmental enrichment alters the basal and nicotine-mediated pERK1/2, which may contribute to enrichment-induced behavioral alterations in response to nicotine. PMID:25328101

  10. The role of ERK-1/2 in the N/OFQ-induced inhibition of delayed rectifier potassium currents.

    PubMed

    Wang, Wei; Cui, Qingbo; Li, Yurong; Li, Baoxin; Yang, Xu; Cui, Lanwei; Jin, Hongbo; Qu, Lihui

    2010-04-16

    Nociceptin/orphanin FQ (N/OFQ) is an endogenous opioid-like heptadecapeptide involved in many neurocognitive functions, including learning and memory. Our previous report showed that N/OFQ inhibits the delayed rectifier potassium current (I(K)), and this effect is associated with protein kinase C (PKC) activation. Therefore, we wanted to determine if extracellular signal-regulated kinase-1/2 (ERK-1/2) signaling is regulated by N/OFQ and associated with the effect of N/OFQ on the I(K). In the current study, we tested if N/OFQ and two PKC activators [phorbol 12,13-dibutyrate (PDBu) and ingenol 3,20-dibenzoate (IDB)] affected the phosphorylation level of ERK-1/2 and its nuclear substrate, ETS-like transcription factor-1 (Elk-1), using western blots. In addition, we tested if ERK-1/2 affected the N/OFQ-induced inhibition of the I(K) by using whole-cell patch-clamp recordings in acutely dissociated rat parietal cortical neurons. We found that N/OFQ, PDBu, and IDB increased the amount of phosphorylated ERK-1/2 and Elk-1; U0126, a specific inhibitor for ERK-1/2, attenuated the inhibitory effect of N/OFQ on the I(K). These data suggest that the ERK-1/2 pathway, at least in part, mediates the inhibitory effect of N/OFQ on the I(K) in acutely dissociated rat cerebral parietal cortical neurons.

  11. Neuronal Differentiation Dictates Estrogen-Dependent Survival and ERK1/2 Kinetic by Means of Caveolin-1

    PubMed Central

    Volpicelli, Floriana; Caiazzo, Massimiliano; Moncharmont, Bruno; di Porzio, Umberto; Colucci-D’Amato, Luca

    2014-01-01

    Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival. PMID:25350132

  12. ERK1/2 signaling plays an important role in topoisomerase II poison-induced G2/M checkpoint activation.

    PubMed

    Kolb, Ryan H; Greer, Patrick M; Cao, Phu T; Cowan, Kenneth H; Yan, Ying

    2012-01-01

    Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-7 and T47D breast cancer cells with topo II poisons resulted in an increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and an subsequent induction of G2/M cell cycle arrest. Furthermore, inhibition of ERK1/2 activation using specific inhibitors markedly attenuated the topo II poison-induced G2/M arrest and diminished the topo II poison-induced activation of ATR and Chk1 kinases. Moreover, decreased expression of ATR by specific shRNA diminished topo II poison-induced G2/M arrest but had no effect on topo II poison-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling had little, if any, effect on topo II poison-induced ATM activation. In addition, ATM inhibition by either incubation of cells with ATM specific inhibitor or transfection of cells with ATM specific siRNA did not block topo II poison-induced G2/M arrest. Ultimately, inhibition of ERK1/2 signaling greatly enhanced topo II poison-induced apoptosis. These results implicate a critical role for ERK1/2 signaling in the activation of G2/M checkpoint response following topo II poison treatment, which protects cells from topo II poison-induced apoptosis.

  13. The role of ERK-1/2 in the N/OFQ-induced inhibition of delayed rectifier potassium currents

    SciTech Connect

    Wang, Wei; Cui, Qingbo; Li, Yurong; Li, Baoxin; Yang, Xu; Cui, Lanwei; Jin, Hongbo; Qu, Lihui

    2010-04-16

    Nociceptin/orphanin FQ (N/OFQ) is an endogenous opioid-like heptadecapeptide involved in many neurocognitive functions, including learning and memory. Our previous report showed that N/OFQ inhibits the delayed rectifier potassium current (I{sub K}), and this effect is associated with protein kinase C (PKC) activation. Therefore, we wanted to determine if extracellular signal-regulated kinase-1/2 (ERK-1/2) signaling is regulated by N/OFQ and associated with the effect of N/OFQ on the I{sub K}. In the current study, we tested if N/OFQ and two PKC activators [phorbol 12,13-dibutyrate (PDBu) and ingenol 3,20-dibenzoate (IDB)] affected the phosphorylation level of ERK-1/2 and its nuclear substrate, ETS-like transcription factor-1 (Elk-1), using western blots. In addition, we tested if ERK-1/2 affected the N/OFQ-induced inhibition of the I{sub K} by using whole-cell patch-clamp recordings in acutely dissociated rat parietal cortical neurons. We found that N/OFQ, PDBu, and IDB increased the amount of phosphorylated ERK-1/2 and Elk-1; U0126, a specific inhibitor for ERK-1/2, attenuated the inhibitory effect of N/OFQ on the I{sub K}. These data suggest that the ERK-1/2 pathway, at least in part, mediates the inhibitory effect of N/OFQ on the I{sub K} in acutely dissociated rat cerebral parietal cortical neurons.

  14. ERK1/ERK2 MAPK signaling is required to increase myelin thickness independent of oligodendrocyte differentiation and initiation of myelination.

    PubMed

    Ishii, Akihiro; Fyffe-Maricich, Sharyl L; Furusho, Miki; Miller, Robert H; Bansal, Rashmi

    2012-06-27

    Wrapping of the myelin sheath around axons by oligodendrocytes is critical for the rapid conduction of electrical signals required for the normal functioning of the CNS. Myelination is a multistep process where oligodendrocytes progress through a well coordinated differentiation program regulated by multiple extracellular growth and differentiation signals. The intracellular transduction of the extracellular signals that regulate myelination is poorly understood. Here we demonstrate a critical role for two important signaling molecules, extracelluar signal-regulated protein kinases 1 and 2 (ERK1/ERK2), downstream mediators of mitogen-activated protein kinases, in the control of CNS myelin thickness. We generated and analyzed two lines of mice lacking both ERK1/ERK2 function specifically in oligodendrocyte-lineage cells. In the absence of ERK1/ERK2 signaling NG2⁺ oligodendrocyte progenitor cells proliferated and differentiated on schedule. Mutant oligodendrocytes also ensheathed axons normally and made a few wraps of compact myelin. However, the subsequent increase in myelination that correlated myelin thickness in proportion to the axon caliber failed to occur. Furthermore, although the numbers of differentiated oligodendrocytes in the adult mutants were unchanged, they showed an inability to upregulate the transcription of major myelin genes that normally occurs during active myelination. Similarly, in vitro ERK1/ERK2-deficient oligodendrocytes differentiated normally but failed to form typical myelin-like membrane sheets. None of these effects were observed in single ERK1 or ERK2 mutants. These studies suggest that the predominant role of ERK1/ERK2 signaling in vivo is in promoting rapid myelin growth to increase its thickness, subsequent to oligodendrocyte differentiation and the initiation of myelination.

  15. ERK1/2 activation modulates pyocyanin-induced toxicity in A549 respiratory epithelial cells.

    PubMed

    Forbes, Amanda; Davey, Andrew K; Perkins, Anthony V; Grant, Gary D; McFarland, Amelia J; McDermott, Catherine M; Anoopkumar-Dukie, Shailendra

    2014-02-01

    Pyocyanin (PCN), a virulence factor produced by Pseudomonas aeruginosa, has many damaging effects on mammalian cells. Several lines of evidence suggest that this damage is primarily mediated by its ability to generate oxidative stress. However mechanisms underlying PCN-induced oxidative injury remain unclear. Although oxidative stress and subsequent MAPK signaling has been shown to modulate cell death in other models, its role in PCN-induced cytotoxicity remains unknown. Therefore the aim of this study was to investigate the role of redox-sensitive MAPK in PCN-induced toxicity in A549 cells. Here we show that PCN (50μM) rapidly increased ERK1/2 phosphorylation after 5min. Pre-treatment of A549 cells with the MEK1/2 inhibitor U0126 (10μM) decreased PCN-induced ERK1/2 phosphorylation and protected cells against apoptosis and cell injury suggesting a role for ERK signalling. In contrast, JNK and p38 MAPK phosphorylation remained unchanged following exposure to PCN and pretreatment with either the JNK or p38 MAPK inhibitors (10μM SP600125 and 10μM SB203580, respectively) did not afford protection against PCN toxicity. This would suggest that PCN-induced cytotoxicity appears to occur independently of JNK and p38 MAPK signaling pathways. Finally, although we confirm that oxidative stress contributes to PCN-induced toxicity, our data suggest the contribution of oxidative stress is independent of ERK1/2 signaling. These findings may provide insight for novel targeted therapies to reduce PCN-mediated lung injury in patients with chronic P. aeruginosa respiratory infections.

  16. Elevated Pressure Enhanced Trail-Induced Apoptosis in Hepatocellular Carcinoma Cells Via Erk1/2-Inactivation.

    PubMed

    Hong, Eunyoung; Lee, Eunil; Kim, Joonhee; Kwon, Daeho; Lim, Yongchul

    2015-12-01

    The high frequency of intrinsic resistance to TNF-related apoptosisinducing ligand (TRAIL) in tumor cell lines has necessitated the development of strategies to sensitize tumors to TRAIL-induced apoptosis. We previously showed that elevated pressure applied as a mechanical stressor enhanced TRAIL-mediated apoptosis in human lung carcinoma cells in vitro and in vivo. This study focused on the effect of elevated pressure on the sensitization of TRAIL-resistant cells and the underlying mechanism. We observed elevated pressure-induced sensitization to TRAIL-mediated apoptosis in Hep3B cells, accompanied by the activation of several caspases and the mitochondrial signaling pathway. Interestingly, the enhanced apoptosis induced by elevated pressure was correlated with suppression of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation and CREB without any change to other MAPKs. Phosphorylation of Bcl-2-associated death promoter (BAD) also decreased, leading to inhibition of the mitochondrial pathway. To confirm whether the activation of pERK1/2 plays a key role in the TRAIL-sensitizing effect of elevated pressure, Hep3B cells were pre-treated with the ERK1/2-specific inhibitor PD98059 instead of elevated pressure. Co-treatment with PD98059 and TRAIL augmented TRAIL-induced apoptosis and decreased BAD phosphorylation. The inhibition of ERK1/2 activation by elevated pressure and PD98059 also reduced BH3 interacting-domain death agonist (BID), thereby amplifying apoptotic stress at the mitochondrial level. Our results suggest that elevated pressure enhances TRAIL-induced apoptosis of Hep3B cells via specific suppression of ERK1/2 activation among MAPKs.

  17. Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling.

    PubMed

    Vietti, Giulia; Ibouraadaten, Saloua; Palmai-Pallag, Mihaly; Yakoub, Yousof; Piret, Jean-Pascal; Marbaix, Etienne; Lison, Dominique; van den Brule, Sybille

    2016-01-01

    Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-β or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-β in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-β- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.

  18. Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

    SciTech Connect

    Luo, Di-xian; Cheng, Jiming; Xiong, Yan; Li, Junmo; Xia, Chenglai; Xu, Canxin; Wang, Chun; Zhu, Bingyang; Hu, Zhuowei; Liao, Duan-fang

    2010-01-22

    Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.

  19. Acquired Cell-Mediated Immunodepression in Acute Chagas' Disease

    PubMed Central

    Teixeira, Antonio R. L.; Teixeira, Glória; Macêdo, Vanize; Prata, Aluizio

    1978-01-01

    In this study two groups of patients with acute Chagas' disease were identified. Group one consisted of five patients with apparent acute Chagas' disease. These patients showed symptoms and signals of an acute illness, such as high fever and enlarged spleen. One of these patients developed severe myocarditis and heart failure. Group two consisted of seven patients with inapparent acute Chagas' disease. This was a nonclinical entity, not perceived by the patient who did not seek medical care. The diagnosis was made by the shift of a serologic test which indicates the presence of immunoglobulin M antibodies to Trypanosoma cruzi. The patients with apparent acute Chagas' disease showed positive delayed-type skin response to T. cruzi antigen. Also, their leukocytes showed significant inhibition of migration in the presence of this antigen. By contrast, the patients with the inapparent acute Chagas' disease did not show positive delayed-type skin response to T. cruzi antigen and no significant inhibition was observed when their cells migrated in the presence of this antigen. Of interest, none of these patients was capable of developing contact sensitivity to 2,4-dinitrochlorobenzene. However, three out of five patients with the apparent acute disease and all the normal control subjects showed positive contact reaction after sensitization to this drug. The results of these experiments would suggest that the thymus-derived (T)-lymphocyte function is depressed in patients with the clinically inapparent acute Chagas' disease. This immunodepression seems to be acquired in the course of the T. cruzi infection because all patients showed positive delayed-type skin response to at least one ubiquitous microbial extract, thus indicating previously normal T-cell function. We hypothesize that T. cruzi antigens may directly stimulate T cells with the concomitant release of factors that might become supressive for T-cell responses. Furthermore, the suppressive effect might interfere

  20. Chlorpyrifos inhibits cell proliferation through ERK1/2 phosphorylation in breast cancer cell lines.

    PubMed

    Ventura, Clara; Venturino, Andrés; Miret, Noelia; Randi, Andrea; Rivera, Elena; Núñez, Mariel; Cocca, Claudia

    2015-02-01

    It is well known the participation of oxidative stress in the induction and development of different pathologies including cancer, diabetes, neurodegeneration and respiratory disorders among others. It has been reported that oxidative stress may be induced by pesticides and it could be the cause of health alteration mediated by pollutants exposure. Large number of registered products containing chlorpyrifos (CPF) is used to control pest worldwide. We have previously reported that 50 μM CPF induces ROS generation and produces cell cycle arrest followed by cell death. The present investigation was designed to identify the pathway involved in CPF-inhibited cell proliferation in MCF-7 and MDA-MB-231 breast cancer cell lines. In addition, we determined if CPF-induced oxidative stress is related to alterations in antioxidant defense system. Finally we studied the molecular mechanisms underlying in the cell proliferation inhibition produced by the pesticide. In this study we demonstrate that CPF (50 μM) induces redox imbalance altering the antioxidant defense system in breast cancer cells. Furthermore, we found that the main mechanism involved in the inhibition of cell proliferation induced by CPF is an increment of p-ERK1/2 levels mediated by H2O2 in breast cancer cells. As PD98059 could not abolish the increment of ROS induced by CPF, we concluded that ERK1/2 phosphorylation is subsequent to ROS production induced by CPF but not the inverse. PMID:25180937

  1. Galla rhois exerts its antiplatelet effect by suppressing ERK1/2 and PLCβ phosphorylation.

    PubMed

    Lee, Jung-Jin; Cho, Won-Kyung; Kwon, Hyeeun; Gu, Minjung; Ma, Jin Yeul

    2014-07-01

    Galla rhois and its components have various biological activities, including protective effects on liver cells as well as antimetastatic, antiplatelet, and antibacterial effects. In the present study, we identified the antiplatelet activity and possible mechanism of action of a G. rhois extract (GRE). We investigated the effect of GRE and its components on rabbit platelet activation, and their possible molecular mechanisms. The GRE inhibited collagen-, AA-, and thrombin-induced platelet aggregation as well as serotonin secretion, in a concentration-dependent manner. The GRE significantly inhibited the production of lipoxygenase-mediated 12-hydroxyeicosatetraenoic acid. The GRE effectively suppressed thrombin-stimulated PLCβ3 phosphorylation and collagen-induced ERK1/2 phosphorylation, in addition, the GRE significantly restored the cAMP level, which had decreased due to collagen or thrombin. Among the components of GRE, methyl gallate inhibited the collagen-induced platelet activation through suppression of ERK phosphorylation, penta-O-galloyl-β-D-glucoside inhibited the thrombin-induced platelet activation through suppression of PLCβ phosphorylation. These results indicate that the GRE including methyl gallate and penta-O-galloyl-β-D-glucoside suppressed platelet activation by inhibiting ERK1/2 and PLCβ3 phosphorylation.

  2. ERK1-Based Pathway as a New Selective Mechanism To Modulate CCR5 with Natural Antibodies.

    PubMed

    Venuti, Assunta; Pastori, Claudia; Siracusano, Gabriel; Riva, Agostino; Sciortino, Maria Teresa; Lopalco, Lucia

    2015-10-01

    Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-lasting internalization (48 h) of the protein, whereas all known CCR5 modulating molecules show a short-term kinetics (60-90 min). Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto unrecognized mechanism of CCR5 modulation mediated by G protein-dependent ERK1 activity. We further demonstrate that ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such as the use of recombinant Abs.

  3. Ifenprodil attenuates the acquisition and expression of methamphetamine-induced behavioral sensitization and activation of Ras-ERK1/2 cascade in the caudate putamen.

    PubMed

    Li, Lu; Qiao, Chuchu; Chen, Gang; Qian, Hongyan; Hou, Ying; Li, Tao; Liu, Xinshe

    2016-10-29

    Chronic discontinuous use of many psychomotor stimulants leads to behavioral sensitization and, owing to it shares common mechanisms with relapse, most researchers use its animal model to explore the neurobiological mechanisms of addiction. Recent studies have proved that N-methyl-d-aspartate receptors (NMDARs) are implicated in psychomotor stimulant-induced behavioral sensitization. However, the function of GluN2B-containing NMDARs and their potential downstream cascade(s) in the acquisition and expression of behavioral sensitization to methamphetamine (METH) have not been explored. In this study, 2.5, 5, and 10mg/kg ifenprodil, the specific inhibitor of GluN2B, was used to explore the function of these receptors in distinct phases of behavioral sensitization to METH in mice. Then, using western blot, Ras, pERK1/2/ERK1/2, and ΔFosB levels in the prefrontal cortex (PFc), nucleus accumbens (NAc), and caudate putamen (CPu) were detected. Behavioral results showed that low-dose ifenprodil attenuated the acquisition and expression of behavioral sensitization to METH significantly. Western blot analysis revealed that pre-injection of low-dose ifenprodil in the acquisition markedly attenuated METH-induced ascent of Ras, pERK1/2/ERK1/2, and ΔFosB protein levels in the CPu. However, pre-treatment in the expression only affected the alterations of Ras and pERK1/2/ERK1/2 levels in the CPu. Moreover, chronic METH administration increased pERK1/2/ERK1/2 level in the NAc. In conclusion, GluN2B-containing NMDARs contribute to both the acquisition and expression of behavioral sensitization to METH in mice. Furthermore, the acquisition phase might be mediated by the Ras-ERK1/2-ΔFosB cascade in the CPu while the expression phase may be regulated by the Ras-ERK1/2 cascade in the CPu. PMID:27544406

  4. Hydroxychloroquine Protects against Cardiac Ischaemia/Reperfusion Injury In Vivo via Enhancement of ERK1/2 Phosphorylation

    PubMed Central

    Bourke, Lauren; McCormick, James; Taylor, Valerie; Pericleous, Charis; Blanchet, Benoit; Costedoat-Chalumeau, Nathalie; Stuckey, Daniel; Lythgoe, Mark F.; Stephanou, Anastasis; Ioannou, Yiannis

    2015-01-01

    An increasing number of investigations including human studies demonstrate that pharmacological ischaemic preconditioning is a viable way to protect the heart from myocardial ischaemia/reperfusion (I/R) injury. This study investigated the role of hydroxychloroquine (HCQ) in the heart during I/R injury. In vitro and in vivo models of myocardial I/R injury were used to assess the effects of HCQ. It was found that HCQ was protective in neonatal rat cardiomyocytes through inhibition of apoptosis, measured by TUNEL and cleaved caspase-3. This protection in vitro was mediated through enhancement of ERK1/2 phosphorylation mediated by HCQ in a dose-dependent fashion. A decrease in infarct size was observed in an in vivo model of myocardial I/R injury in HCQ treated animals and furthermore this protection was blocked in the presence of the ERK1/2 inhibitor U0126. For the first time, we have shown that HCQ promotes a preconditioning like protection in an in vivo simulated rat myocardial I/R injury model. Moreover, it was shown that HCQ is protective via enhanced phosphorylation of the pro-survival kinase ERK1/2. PMID:26636577

  5. Multiple transport systems mediate virus-induced acquired resistance to oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this paper, we report the phenomenon of acquired cross-tolerance to oxidative (UV-C and H2O2) stress in Nicotiana benthamiana plants infected with Potato virus X (PVX) and investigate the functional expression of transport systems in mediating this phenomenon. By combining multiple approaches, we...

  6. Protocatechuic Acid from Alpinia oxyphylla Induces Schwann Cell Migration via ERK1/2, JNK and p38 Activation.

    PubMed

    Ju, Da-Tong; Kuo, Wei-Wen; Ho, Tsung-Jung; Paul, Catherine Reena; Kuo, Chia-Hua; Viswanadha, Vijaya Padma; Lin, Chien-Chung; Chen, Yueh-Sheng; Chang, Yung-Ming; Huang, Chih-Yang

    2015-01-01

    Alpinia oxyphylla MIQ (Alpinate Oxyphyllae Fructus, AOF) is an important traditional Chinese medicinal herb whose fruits is widely used to prepare tonics and is used as an aphrodisiac, anti salivary, anti diuretic and nerve-protective agent. Protocatechuic acid (PCA), a simple phenolic compound was isolated from the kernels of AOF. This study investigated the role of PCA in promoting neural regeneration and the underlying molecular mechanisms. Nerve regeneration is a complex physiological response that takes place after injury. Schwann cells play a crucial role in the endogenous repair of peripheral nerves due to their ability to proliferate and migrate. The role of PCA in Schwann cell migration was determined by assessing the induced migration potential of RSC96 Schwann cells. PCA induced changes in the expression of proteins of three MAPK pathways, as determined using Western blot analysis. In order to determine the roles of MAPK (ERK1/2, JNK, and p38) pathways in PCA-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production, the expression of several MAPK-associated proteins was analyzed after siRNA-mediated inhibition assays. Treatment with PCA-induced ERK1/2, JNK, and p38 phosphorylation that activated the downstream expression of PAs and MMPs. PCA-stimulated ERK1/2, JNK and p38 phosphorylation was attenuated by individual pretreatment with siRNAs or MAPK inhibitors (U0126, SP600125, and SB203580), resulting in the inhibition of migration and the uPA-related signal pathway. Taken together, our data suggest that PCA extract regulate the MAPK (ERK1/2, JNK, and p38)/PA (uPA, tPA)/MMP (MMP2, MMP9) mediated regeneration and migration signaling pathways in Schwann cells. Therefore, PCA plays a major role in Schwann cell migration and the regeneration of damaged peripheral nerve.

  7. Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain.

    PubMed

    Zhang, Yan-Bo; Guo, Zheng-Dong; Li, Mei-Yi; Fong, Peter; Zhang, Ji-Guo; Zhang, Can-Wen; Gong, Ke-Rui; Yang, Ming-Feng; Niu, Jing-Zhong; Ji, Xun-Ming; Lv, Guo-Wei

    2015-01-01

    Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6-S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our

  8. Simvastatin induces differentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways.

    PubMed

    Han, Yohan; Kim, Song Ja

    2016-08-15

    Statins are competitive inhibitors of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, a key enzyme involved in the conversion of HMG-CoA to the cholesterol precursor mevalonate. Some statins, such as simvastatin (simvastatin), have been shown to have anti-cancer and anti-inflammatory effects, reducing cartilage degradation in osteoarthritic rabbits in vivo. However, the regulatory mechanisms undergirding simvastatin mediated chondrocyte differentiation have not been well elucidated. Thus, we investigated the action and mechanism of simvastatin on differentiation of rabbit articular chondrocytes through western blot analyses, RT-PCR, and immunohistochemical (IHC) and immunofluorescence (IF) staining. Simvastatin treatment was found to induce type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. Indeed, RT-PCR revealed increased expression of type II collagen on treatment with simvastatin. Both IHC and IF staining indicated differentiation of chondrocytes. Simvastatin treatment reduced activation of ERK-1/2 and stimulated activation of p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced simvastatin induced differentiation, whereas inhibition of p38 kinase with SB203580 inhibited simvastatin induced differentiation. Simvastatin treatment also inhibits loss of type II collagen in serial monolayer culture. Collectively, our results indicate that ERK-1/2 and p38 kinase regulate simvastatin-induced differentiation of chondrocytes in opposing manners. Thus, these findings suggest that simvastatin may be a potential therapeutic drug for osteoarthritis.

  9. AG and UAG induce β-casein expression via activation of ERK1/2 and AKT pathways

    PubMed Central

    Li, Sunan; Liu, Juxiong; Lv, Qingkang; Zhang, Chuan; Xu, Shiyao; Yang, Dongxue; Huang, Bingxu; Zeng, Yalong; Gao, Yingjie

    2016-01-01

    Abstract The ghrelin peptides were found to circulate in two major forms: acylated ghrelin (AG) and unacylated ghrelin (UAG). Previous studies showed that AG regulates β-casein (CSN2) expression in mammary epithelial cells. However, little is known about the mechanisms by which AG regulates CSN2 gene and protein expression. Evidence suggests that UAG has biological activity through GHSR1a-independent mechanisms. Here, we investigated the possible GHSR1a-mediated effect of UAG on the expression of CSN2 in primary bovine mammary epithelial cells (pbMECs) isolated from lactating cow. We found that both AG and UAG increase the expression of CSN2 in a dose-dependent manner in pbMECs in comparison with the control group. Increased expression of CSN2 was blocked by [D-Lys3]-GHRP-6 (an antagonist of the GHSR1a) and NF449 (a Gs-α subunit inhibitor) in pbMECs. In addition, both AG and UAG activated AKT/protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, whereas [D-Lys3]-GHRP-6 and NF449 inhibited the phosphorylation of AKT and ERK1/2 in pbMECs respectively. Blockade of ERK1/2 and AKT signaling pathways prevented the expression of CSN2 induced by AG or UAG. Finally, we found that both AG and UAG cause cell proliferation through identical signaling pathways. Taken together, these results demonstrate that both AG and UAG act on ERK1/2 and AKT signaling pathways to facilitate the expression of CSN2 in a GHSR1a-dependent manner. PMID:26873999

  10. Arsenite induces cell transformation by reactive oxygen species, AKT, ERK1/2, and p70S6K1

    SciTech Connect

    Carpenter, Richard L.; Jiang, Yue; Jing, Yi; He, Jun; Rojanasakul, Yon; Liu, Ling-Zhi; Jiang, Bing-Hua

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer Chronic exposure to arsenite induces cell proliferation and transformation. Black-Right-Pointing-Pointer Arsenite-induced transformation increases ROS production and downstream signalings. Black-Right-Pointing-Pointer Inhibition of ROS levels via catalase reduces arsenite-induced cell transformation. Black-Right-Pointing-Pointer Interruption of AKT, ERK, or p70S6K1 inhibits arsenite-induced cell transformation. -- Abstract: Arsenic is naturally occurring element that exists in both organic and inorganic formulations. The inorganic form arsenite has a positive association with development of multiple cancer types. There are significant populations throughout the world with high exposure to arsenite via drinking water. Thus, human exposure to arsenic has become a significant public health problem. Recent evidence suggests that reactive oxygen species (ROS) mediate multiple changes to cell behavior after acute arsenic exposure, including activation of proliferative signaling and angiogenesis. However, the role of ROS in mediating cell transformation by chronic arsenic exposure is unknown. We found that cells chronically exposed to sodium arsenite increased proliferation and gained anchorage-independent growth. This cell transformation phenotype required constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. We also observed these cells constitutively produce ROS, which was required for the constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. Suppression of ROS levels by forced expression of catalase also reduced cell proliferation and anchorage-independent growth. These results indicate cell transformation induced by chronic arsenic exposure is mediated by increased cellular levels of ROS, which mediates activation of AKT, ERK1/2, and p70S6K1.

  11. ERK1 nucleocytoplasmic shuttling rate depends on specific N-terminal aminoacids

    SciTech Connect

    Marchi, Matilde; Pancrazi, Laura; Maffei, Margherita; Ratto, G. Michele; Costa, Mario

    2010-07-23

    Despite ERK1 and ERK2 were considered interchangeable isoforms for a long time, their roles are now emerging as only partially overlapping. We recently reported that the nucleocytoplasmic trafficking of GFP-tagged ERK1 is slower than that of ERK2, this difference being caused by a unique domain of ERK1 located at its N-terminus (ERK1-Nt). In the present report we further investigated this issue by asking which were the specific aminoacids involved in such process. By photobleaching strategy, we demonstrated that ERK1-Nt is a domain capable to slow down the nucleocytoplasmic shuttling rate even of a small cargo protein. ERK1-Nt was then dissected into three regions as follows: 1 (aa 1-9), 2 (aa 10-29) and 3, (aa 30-39) that were deleted or mutated at specific sites. Dynamic imaging assessment of the role played by each region in determining the shuttling rate revealed that: region 1 has no significant role, region 2 and specific aminoacids of region 3 (V{sub 31}, K{sub 33,} P{sub 36}) are critical, but singularly do not totally account for the difference in the shuttling rate between ERK1 and 2. Finally, we demonstrated that the nucleocytoplasmic shuttling rate of a passively diffusing protein (mRED) is inversely related to ERK1-Nt-GFP concentrations inside the cell, thus suggesting that ERK1-Nt-GFP occupies the nuclear pore perhaps because of an important affinity of ERK1-Nt for nucleoporins. In conclusion, ERK1-Nt is a domain able per se to confer a slower shuttling rate to a cargo protein. Specific regions within this domain were identified as responsible for this biophysical property.

  12. Signaling pathways of interleukin-1 actions in the brain: anatomical distribution of phospho-ERK1/2 in the brain of rat treated systemically with interleukin-1beta.

    PubMed

    Nadjar, A; Combe, C; Busquet, P; Dantzer, R; Parnet, P

    2005-01-01

    Interleukin-1beta is released at the periphery during infection and acts on the nervous system to induce fever, neuroendocrine activation, and behavioral changes. These effects are mediated by brain type I IL-1 receptors. In vitro studies have shown the ability of interleukin-1beta to activate mitogen-activated protein kinase signaling pathways including p38, c-Jun N-terminal kinase and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In contrast to other mitogen-activated protein kinases, little is known about ERK1/2 activation in the rat brain in response to interleukin-1beta. The aim of the present study was therefore to investigate spatial and temporal activation of ERK1/2 in the rat brain after peripheral administration of interleukin-1beta using immunohistochemistry to detect the phosphorylated form of the kinase. In non-stimulated conditions, phosphorylated ERK1/2 immunoreactivity was observed in neurons throughout the brain. Administration of interleukin-1beta (60 microg/kg, i.p.) induced the phosphorylation of ERK1/2 in areas at the interface between brain and blood or cerebrospinal fluid: meninges, circumventricular organs, endothelial like cells of the blood vessels, and in brain nuclei involved in behavioral depression, fever and neuroendocrine activation: paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala and arcuate nucleus. Double labeling of phosphorylated ERK1/2 and cell markers revealed the expression of phosphorylated ERK1/2 in neurons, astrocytes and microglia. Since phosphorylated ERK1/2 was found in structures in which type I IL-1 receptor has already been identified as well as in structures lacking this receptor, activation of ERK1/2 is likely to occur in response to both direct and indirect action of interleukin-1beta on its target cells.

  13. (-)-Epigallocatechin gallate suppresses proliferation of vascular smooth muscle cells induced by high glucose by inhibition of PKC and ERK1/2 signalings.

    PubMed

    Yang, Jian; Han, Yu; Sun, Hailan; Chen, Caiyu; He, Duofen; Guo, Jing; Yu, Changqing; Jiang, Baoquan; Zhou, Lin; Zeng, Chunyu

    2011-11-01

    Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetes-related vascular complications. (-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, is able to exert antidiabetes effects in animal models. However, it is not known whether or not EGCG inhibits VSMC proliferation induced by high glucose. This study tested the hypothesis that EGCG might have an inhibitory effect on VSMC proliferation induced by high glucose. VSMC proliferation was determined by [(3)H]-thymidine incorporation and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by immunoblotting, and ERK 1/2 activity was detected by measuring the ability to phosphorylate its substrate Elk-1. Glucose increased VSMC proliferation in a concentration-dependent manner, which was reduced in the presence of EGCG. VSMC proliferation mediated by high glucose (30 mM) was involved in protein kinase C (PKC) and ERK1/2 signalings, because its effect was blocked by PKC inhibitor (PKC inhibitor 19-31) and ERK1/2 inhibitor (PD98059). Pretreatment of VSMCs with EGCG significantly inhibited the stimulatory effect of high glucose on PKC and ERK1/2 activation, followed by attenuation of its downstream transcription factor Elk-1 phosphorylation. Taken together, these results suggest that EGCG could suppress VSMC proliferation induced by high glucose by inhibition of PKC and ERK1/2 signalings in VSMCs, which indicates that EGCG might be a possible medicine to reduce vascular complications in diabetes.

  14. miR-155 Inhibits Nucleus Pulposus Cells' Degeneration through Targeting ERK 1/2.

    PubMed

    Ye, Dongping; Dai, Libing; Yao, Yicun; Qin, Shengnan; Xie, Han; Wang, Wen; Liang, Weiguo

    2016-01-01

    We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2. PMID:27635110

  15. miR-155 Inhibits Nucleus Pulposus Cells' Degeneration through Targeting ERK 1/2

    PubMed Central

    Dai, Libing; Yao, Yicun; Qin, Shengnan; Xie, Han; Wang, Wen

    2016-01-01

    We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2. PMID:27635110

  16. miR-155 Inhibits Nucleus Pulposus Cells' Degeneration through Targeting ERK 1/2

    PubMed Central

    Dai, Libing; Yao, Yicun; Qin, Shengnan; Xie, Han; Wang, Wen

    2016-01-01

    We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2.

  17. Both ERK1 and ERK2 are required for enterovirus 71 (EV71) efficient replication.

    PubMed

    Zhu, Meng; Duan, Hao; Gao, Meng; Zhang, Hao; Peng, Yihong

    2015-03-01

    It has been demonstrated that MEK1, one of the two MEK isoforms in Raf-MEK-ERK1/2 pathway, is essential for successful EV71 propagation. However, the distinct function of ERK1 and ERK2 isoforms, the downstream kinases of MEKs, remains unclear in EV71 replication. In this study, specific ERK siRNAs and selective inhibitor U0126 were applied. Silencing specific ERK did not significantly impact on the EV71-caused biphasic activation of the other ERK isoform, suggesting the EV71-induced activations of ERK1 and ERK2 were non-discriminative and independent to one another. Knockdown of either ERK1 or ERK2 markedly impaired progeny EV71 propagation (both by more than 90%), progeny viral RNA amplification (either by about 30% to 40%) and protein synthesis (both by around 70%), indicating both ERK1 and ERK2 were critical and not interchangeable to EV71 propagation. Moreover, suppression of EV71 replication by inhibiting both early and late phases of ERK1/2 activation showed no significant difference from that of only blocking the late phase, supporting the late phase activation was more importantly responsible for EV71 life cycle. Taken together, this study for the first time identified both ERK1 and ERK2 were required for EV71 efficient replication and further verified the important role of MEK1-ERK1/2 in EV71 replication.

  18. Regulation of ERK1/2 activity upon contact inhibition in fibroblasts

    SciTech Connect

    Kueppers, Monika; Faust, Dagmar; Linz, Berenike; Dietrich, Cornelia

    2011-03-18

    Research highlights: {yields} Regulation of ERK1/2 activity upon contact inhibition was investigated. {yields} Upstream activation of ERK is attenuated upon contact inhibition. {yields} ERK phosphatases are probably not involved in ERK1/2 dephosphorylation. {yields} Signaling of the EGFR and PDGFR is differentially inhibited upon contact inhibition. -- Abstract: Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Despite its generally accepted importance for maintaining tissue homeostasis knowledge about the underlying molecular mechanisms of contact inhibition is still scarce. Since the MAPK ERK1/2 plays a pivotal role in the control of proliferation, we investigated regulation of ERK1/2 phosphorylation which is downregulated in confluent NIH3T3 cultures. We found a decrease in upstream signaling including phosphorylation of the growth factor receptor adaptor protein ShcA and the MAPK kinase MEK1/2 in confluent compared to exponentially growing cultures whereas involvement of ERK1/2 phosphatases in ERK1/2 inactivation is unlikely. Treatment of confluent, serum-deprived cultures with PDGF-B resulted in similar phosphorylation of ERK1/2 and induction of DNA-synthesis as detected in sparse, serum-deprived cultures. In contrast, ERK1/2 phosphorylation and DNA-synthesis could not be stimulated in confluent, serum-deprived cultures exposed to EGF. Our data indicate that PDGFR- and EGFR signaling are differentially inhibited in confluent cultures of NIH3T3 cells.

  19. Decoy receptor 3 suppresses FasL-induced apoptosis via ERK1/2 activation in pancreatic cancer cells

    SciTech Connect

    Zhang, Yi; Li, Dechun; Zhao, Xin; Song, Shiduo; Zhang, Lifeng; Zhu, Dongming; Wang, Zhenxin; Chen, Xiaochen; Zhou, Jian

    2015-08-07

    Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level of DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ{sup 2} = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26  ±  2.67%) was significantly higher than that of DcR3 negative specimens (43.58  ±  7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells. - Highlights: • We investigated the role of DcR3 in FasL resistance in pancreatic cancer. • Knockdown of DcR3 in SW1990 cells reduced resistance to FasL-induced apoptosis. • DcR3 knockdown also elevated caspase expression, and reduced ERK1/2 phosphorylation. • Tumor and non-tumor tissues were collected from 66 pancreatic carcinoma patients

  20. Receptor sequestration in response to β-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression

    PubMed Central

    Paradis, Justine S.; Ly, Stevenson; Blondel-Tepaz, Élodie; Galan, Jacob A.; Beautrait, Alexandre; Scott, Mark G. H.; Enslen, Hervé; Marullo, Stefano; Roux, Philippe P.; Bouvier, Michel

    2015-01-01

    MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and β-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with β-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with β-arrestin, implicating this scaffolding protein in the receptor’s subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking β-arrestins combined with in vitro kinase assays revealed that β-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator. PMID:26324936

  1. The PHA Test Reflects Acquired T-Cell Mediated Immunocompetence in Birds

    PubMed Central

    Tella, José L.; Lemus, Jesús A.; Carrete, Martina; Blanco, Guillermo

    2008-01-01

    Background cological immunology requires techniques to reliably measure immunocompetence in wild vertebrates. The PHA-skin test, involving subcutaneous injection of a mitogen (phytohemagglutinin, PHA) and measurement of subsequent swelling as a surrogate of T-cell mediated immunocompetence, has been the test of choice due to its practicality and ease of use in the field. However, mechanisms involved in local immunological and inflammatory processes provoked by PHA are poorly known, and its use and interpretation as an acquired immune response is currently debated. Methodology Here, we present experimental work using a variety of parrot species, to ascertain whether PHA exposure produces larger secondary than primary responses as expected if the test reflects acquired immunocompetence. Moreover, we simultaneously quantified T-lymphocyte subsets (CD4+, CD5+ and CD8+) and plasma proteins circulating in the bloodstream, potentially involved in the immunological and inflammatory processes, through flow cytometry and electrophoresis. Principal Findings Our results showed stronger responses after a second PHA injection, independent of species, time elapsed and changes in body mass of birds between first and second injections, thus supporting the adaptive nature of this immune response. Furthermore, the concomitant changes in the plasma concentrations of T-lymphocyte subsets and globulins indicate a causal link between the activation of the T-cell mediated immune system and local tissue swelling. Conclusions/Significance These findings justify the widespread use of the PHA-skin test as a reliable evaluator of acquired T-cell mediated immunocompetence in diverse biological disciplines. Further experimental research should be aimed at evaluating the relative role of innate immunocompetence in wild conditions, where the access to dietary proteins varies more than in captivity, and to ascertain how PHA responses relate to particular host-parasite interactions. PMID:18820730

  2. ERK 1/2 and PI-3 kinase pathways as a potential mechanism of ghrelin action on cell proliferation and apoptosis in the porcine ovarian follicular cells.

    PubMed

    Rak-Mardyla, A; Gregoraszczuk, E L

    2010-08-01

    Recently, we reported the stimulatory effect of ghrelin on ovarian cell proliferation in parallel with the inhibitory action of ghrelin on cell apoptosis. The aim of the presented data propose local activation of extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2) and phosphoinositide-3 (PI-3) kinase pathways as a mechanism of ghrelin effect in the porcine ovary. To test this hypothesis, action of ghrelin on levels of ERK 1/2 with PI-3 kinase activity and protein expression using ELISA and western blot analysis, respectively, was examined. Additionally, to determine which pathways (ERK 1/2 or PI-3 kinase) are the potential signals of ghrelin-mediated cell proliferation and apoptosis in ovarian cells, we used PD098059 (50 microM) and wortmannin (200 microM), well-known inhibitors of these kinases. Treatment of ovarian coculture cells with ghrelin (100, 250, 500 and 1000 pg/ml) showed stimulation of phospho-ERK 1/2 levels and PI-3 kinase activity, with the maximum effect observed after 15 min of cell incubation. Additionally, western blot analysis indicated that ghrelin increased expression of both kinases. Moreover, ghrelin used in combination with PD098059 or wortmannin significantly decreased cell proliferation, which was measured by the Alamar Blue assay and increased apoptosis, which was measured by caspase - 3 activity and DNA fragmentation. In conclusion, these results suggest that the ERK 1/2 and PI-3 kinase pathways may be potential signals of ghrelin mediate the cell proliferation and apoptosis of ovary cells.

  3. Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis

    PubMed Central

    2012-01-01

    Background Ascites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized. Methods Using human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments. Results In the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression. Conclusions These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells. PMID:23158473

  4. Upregulation of ERK1/2-eNOS via AT2 Receptors Decreases the Contractile Response to Angiotensin II in Resistance Mesenteric Arteries from Obese Rats

    PubMed Central

    Hagihara, Graziela N.; Lobato, Nubia S.; Filgueira, Fernando P.; Akamine, Eliana H.; Aragão, Danielle S.; Casarini, Dulce E.; Carvalho, Maria Helena C.; Fortes, Zuleica B.

    2014-01-01

    It has been clearly established that mitogen-activated protein kinases (MAPKS) are important mediators of angiotensin II (Ang II) signaling via AT1 receptors in the vasculature. However, evidence for a role of these kinases in changes of Ang II-induced vasoconstriction in obesity is still lacking. Here we sought to determine whether vascular MAPKs are differentially activated by Ang II in obese animals. The role of AT2 receptors was also evaluated. Male monosodium glutamate-induced obese (obese) and non-obese Wistar rats (control) were used. The circulating concentrations of Ang I and Ang II, determined by HPLC, were increased in obese rats. Ang II-induced isometric contraction was decreased in endothelium-intact resistance mesenteric arteries from obese compared with control rats and exhibited a retarded AT1 receptor antagonist response. Blocking of AT2 receptors and inhibition of either endothelial nitric oxide synthase (eNOS) or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) restored Ang II-induced contraction in obese rats. Western blot analysis revealed increased protein expression of AT2 receptors in arteries from obese rats. Basal and Ang II-induced ERK1/2 phosphorylation was also increased in obese rats. Blockade of either AT1 or AT2 receptors corrected the increased ERK1/2 phosphorylation in arteries from obese rats to levels observed in control preparations. Phosphorylation of eNOS was increased in obese rats. Incubation with the ERK1/2 inhibitor before Ang II stimulation did not affect eNOS phosphorylation in control rats; however, it corrected the increased phosphorylation of eNOS in obese rats. These results clearly demonstrate that enhanced AT2 receptor and ERK1/2-induced, NO-mediated vasodilation reduces Ang II-induced contraction in an endothelium-dependent manner in obese rats. PMID:25170617

  5. Intra-amygdala inhibition of ERK(1/2) potentiates the discriminative stimulus effects of alcohol.

    PubMed

    Besheer, Joyce; Fisher, Kristen R; Cannady, Reginald; Grondin, Julie J M; Hodge, Clyde W

    2012-03-17

    Extracellular signal-regulated kinase (ERK(1/2)) has been implicated in modulating drug seeking behavior and is a target of alcohol and other drugs of abuse. Given that the discriminative stimulus (subjective/interoceptive) effects of drugs are determinants of abuse liability and can influence drug seeking behavior, we examined the role of ERK(1/2) in modulating the discriminative stimulus effects of alcohol. Using drug discrimination procedures, rats were trained to discriminate a moderate intragastric (IG) alcohol dose (1g/kg) versus water (IG). Following an alcohol (1g/kg) discrimination session phosphorylated ERK(1/2) (pERK(1/2)) immunoreactivity (IR) was significantly elevated in the amygdala, but not the nucleus accumbens. Therefore, we hypothesized that intra-amygdala inhibition of ERK(1/2) would disrupt expression of the discriminative stimulus effects of alcohol. However, intra-amygdala or accumbens administration of the MEK/ERK(1/2) inhibitor U0126 (1 and 3μg) had no effect on the discriminative stimulus effects of the training dose of alcohol (1g/kg). Contrary to our hypothesis, intra-amygdala infusion of U0126 (3μg) potentiated the discriminative stimulus effects of a low alcohol dose (0.5g/kg) and had no effect following nucleus accumbens infusion. Importantly, site-specific inhibition of pERK(1/2) in each brain region was confirmed. Therefore, the increase in pERK(1/2) IR in the amygdala following systemic alcohol administration may be reflective of the widespread effects of alcohol on the brain (activation/inhibition of brain circuits), whereas the site specific microinjection studies confirmed functional involvement of intra-amygdala ERK(1/2). These findings show that activity of the ERK signaling pathway in the amygdala can influence the discriminative stimulus effects of alcohol.

  6. Egr1 is rapidly and transiently induced by estrogen and bisphenol A via activation of nuclear estrogen receptor-dependent ERK1/2 pathway in the uterus.

    PubMed

    Kim, Hye-Ryun; Kim, Yeon Sun; Yoon, Jung Ah; Lyu, Sang Woo; Shin, Hyejin; Lim, Hyunjung J; Hong, Seok-Ho; Lee, Dong Ryul; Song, Haengseok

    2014-12-01

    Coordinate actions of ovarian estrogen (E2) and progesterone (P4) via their own receptors are critical for establishing uterine receptivity for embryo implantation in the uterus. E2 regulates expression of an array of genes to mediate its major actions on heterogeneous uterine cell types. Here we have investigated regulatory mechanism(s) of E2 and bisphenol A (BPA), an endocrine disruptor with potent estrogenic activity on expression of early growth response 1 (Egr1), a zinc finger transcription factor that regulates cell growth, differentiation and apoptosis in the uterus. Egr1 was rapidly and transiently induced by E2 and BPA mainly in stromal cells via nuclear estrogen receptor (ER)-ERK1/2 pathway. ICI 182,780, an ER antagonist, effectively inhibited their actions on EGR1 expression following ERK1/2 phosphorylation. Administration of pharmacological inhibitors for ERK1/2, but not AKT significantly blocked EGR1 expression induced by E2 and BPA. P4 effectively dampened action(s) of E2 and BPA on Egr1 expression via nuclear progesterone receptor. Its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, EGR1 is specifically induced in stromal cells surrounding implanting blastocyst. Collectively, our results show that through nuclear ER-dependent ERK1/2 phosphorylation, not only E2 but also endocrine disruptors with estrogenic activity such as BPA rapidly and transiently induce Egr1 which may be important for embryo implantation and decidualization in mouse uterus.

  7. Kruppel-like factor 2 inhibit the angiogenesis of cultured human liver sinusoidal endothelial cells through the ERK1/2 signaling pathway

    SciTech Connect

    Zeng, Xiao-Qing; Li, Na; Pan, Du-Yi; Miao, Qing; Ma, Gui-Fen; Liu, Yi-Mei; Tseng, Yu-Jen; Li, Feng; Xu, Li-Li; Chen, Shi-Yao

    2015-09-04

    Kruppel-like factor 2 (KLF2) is a crucial anti-angiogenic factor. However, its precise role in hepatic angiogenesis induced by liver sinusoidal endothelial cells (LSECs) remain unclear. This study was aimed to evaluate the effect of KLF2 on angiogenesis of LSECs and to explore the corresponding mechanism. Cultured human LSECs were infected with different lentiviruses to overexpress or suppress KLF2 expression. The CCK-8 assay, transwell migration assay and tube formation test, were used to investigate the roles of KLF2 in the proliferation, migration and vessel tube formation of LSECs, respectively. The expression and phosphorylation of ERK1/2 were detected by western blot. We discovered that the up-regulation of KLF2 expression dramatically inhibited proliferation, migration and tube formation in treated LSECs. Correspondingly, down-regulation of KLF2 expression significantly promoted proliferation, migration and tube formation in treated LSECs. Additionally, KLF2 inhibited the phosphorylation of ERK1/2 pathway, followed by the function of KLF2 in the angiogenesis of LSECs disrupted. In conclusion, KLF2 suppressed the angiogenesis of LSECs through inhibition of cell proliferation, migration, and vessel tube formation. These functions of KLF2 may be mediated through the ERK1/2 signaling pathway. - Highlights: • Overexpression of KLF2 inhibits the proliferation and migration of LSECs. • Overexpression of KLF2 inhibits the angiogenesis of LSECs. • ERK1/2 signaling pathway involved in the anti-angiogenic process of KLF2 on LSECs.

  8. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells

    PubMed Central

    Ji, Yun; Dai, Zhaolai; Wu, Guoyao; Wu, Zhenlong

    2016-01-01

    Excessive reactive oxygen species (ROS) induces oxidative damage to cellular constituents, ultimately leading to induction of apoptotic cell death and the pathogenesis of various diseases. The molecular mechanisms for the action of ROS in intestinal diseases remain poorly defined. Here, we reported that 4-hydroxy-2-nonenal (4-HNE) treatment led to capses-3-dependent apoptosis accompanied by increased intracellular ROS level and reduced glutathione concentration in intestinal epithelial cells. These effects of 4-HNE were markedly abolished by the antioxidant L-cysteine derivative N-acetylcysteine (NAC). Further studies demonstrated that the protective effect of NAC was associated with restoration of intracellular redox state by Nrf2-related regulation of expression of genes involved in intracellular glutathione (GSH) biosynthesis and inactivation of 4-HNE-induced phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). The 4-HNE-induced ERK1/2 activation was mediated by repressing mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of ERK1/2, through a proteasome-dependent degradation mechanism. Importantly, either overexpression of MKP-1 or NAC treatment blocked 4-HNE-induced MKP-1 degradation, thereby protecting cell from apoptosis. These novel findings provide new insights into a functional role of MKP-1 in oxidative stress-induced cell death by regulating ERK1/2 MAP kinase in intestinal epithelial cells. PMID:27620528

  9. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells.

    PubMed

    Ji, Yun; Dai, Zhaolai; Wu, Guoyao; Wu, Zhenlong

    2016-01-01

    Excessive reactive oxygen species (ROS) induces oxidative damage to cellular constituents, ultimately leading to induction of apoptotic cell death and the pathogenesis of various diseases. The molecular mechanisms for the action of ROS in intestinal diseases remain poorly defined. Here, we reported that 4-hydroxy-2-nonenal (4-HNE) treatment led to capses-3-dependent apoptosis accompanied by increased intracellular ROS level and reduced glutathione concentration in intestinal epithelial cells. These effects of 4-HNE were markedly abolished by the antioxidant L-cysteine derivative N-acetylcysteine (NAC). Further studies demonstrated that the protective effect of NAC was associated with restoration of intracellular redox state by Nrf2-related regulation of expression of genes involved in intracellular glutathione (GSH) biosynthesis and inactivation of 4-HNE-induced phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). The 4-HNE-induced ERK1/2 activation was mediated by repressing mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of ERK1/2, through a proteasome-dependent degradation mechanism. Importantly, either overexpression of MKP-1 or NAC treatment blocked 4-HNE-induced MKP-1 degradation, thereby protecting cell from apoptosis. These novel findings provide new insights into a functional role of MKP-1 in oxidative stress-induced cell death by regulating ERK1/2 MAP kinase in intestinal epithelial cells. PMID:27620528

  10. Dose-dependent effect of tamoxifen in tamoxifen-resistant breast cancer cells via stimulation by the ERK1/2 and AKT signaling pathways.

    PubMed

    Wang, Li-Juan; Han, Su-Xia; Bai, E; Zhou, Xia; Li, Meng; Jing, Gui-Hua; Zhao, Jing; Yang, An-Gang; Zhu, Qing

    2013-04-01

    The majority of breast cancers undergo progression from an initially endocrine responsive phenotype to an endocrine therapy-resistant phenotype, and acquired resistance to tamoxifen (Tam) is a major clinical problem. In the present study, we aimed to identify the function and mechanism of Tam at different concentrations in cells with acquired Tam resistance. Estrogen-dependent MCF-7 cells were cultured with Tam to generate Tam-resistant (TAM-R) breast cancer cells or in estrogen-free medium to mimic the effects of clinical treatment. In addition, we analyzed the effects of different concentrations of Tam on TAM-R cells by cell counting. Furthermore, the crosstalk between the stimulatory G protein α subunit (Gαs) and the activation of ERK1/2 and AKT in TAM-R cells was examined by small interfering RNA (siRNA) and immunoblotting methods. Low-dose Tam was found to act as an estrogen agonist via stimulation of the ERK1/2 signaling pathway, resulting in acquired resistance to Tam, whereas high-dose Tam inhibited TAM-R cell growth by blocking the activation of ERK1/2 and AKT. Moreover, Gαs was involved in Tam resistance in breast cancer cells. Taken together, our study demonstrated a dose-dependent growth response to Tam in TAM-R cells, which will promote the understanding of the importance of the appropriate use and dosage of Tam in the clinic.

  11. G-CSF Stimulates Jak2-Dependent Gab2 Phosphorylation Leading to Erk1/2 Activation and Cell Proliferation

    PubMed Central

    Wang, Lin; Xue, Jia; Zadorozny, Eva V.; Robinson, Lisa J.

    2009-01-01

    Granulocyte colony-stimulating factor (G-CSF), the major cytokine regulator of neutrophilic granulopoiesis, stimulates both the proliferation and differentiation of myeloid precursors. A variety of signaling proteins have been identified as mediators of G-CSF signaling, but understanding of their specific interactions and organization into signaling pathways for particular cellular effects is incomplete. The present study examined the role of the scaffolding protein Grb2-associated binding protein-2 (Gab2) in G-CSF signaling. We found that a chemical inhibitor of Janus kinases inhibited G-CSF-stimulated Gab2 phosphorylation. Transfection with Jak2 antisense and dominant negative constructs also inhibited Gab2 phosphorylation in response to G-CSF. In addition, G-CSF enhanced the association of Jak2 with Gab2. In vitro, activated Jak2 directly phosphorylated specific Gab2 tyrosine residues. Mutagenesis studies revealed that Gab2 tyrosine 643 (Y643) was a major target of Jak2 in vitro, and a key residue for Jak2-dependent phosphorylation in intact cells. Mutation of Gab2 Y643 inhibited G-CSF-stimulated Erk1/2 activation and Shp2 binding to Gab2. Loss of Y643 also inhibited Gab2-mediated G-CSF-stimulated cell proliferation. Together, these results identify a novel signaling pathway involving Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation in response to G-CSF. PMID:18644434

  12. Serotonin increases ERK1/2 phosphorylation in astrocytes by stimulation of 5-HT2B and 5-HT2C receptors.

    PubMed

    Li, Baoman; Zhang, Shiquen; Li, Min; Hertz, Leif; Peng, Liang

    2010-11-01

    We have previously shown that fluoxetine causes ERK(1/2) phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT(2B) receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK(1/2) phosphorylation by stimulation of 5-HT(2B) receptors with high affinity (EC(50): 20-30 pM), and by stimulation of 5-HT(2C) receptor with low affinity (EC(50): 1 microM or higher). ERK(1/2) phosphorylation induced by stimulation of either 5-HT(2B) or 5-HT(2C) receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT(2B) receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT(2B)-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT(2B) receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT(2B) receptors. In contrast the relevance of the observed 5-HT(2C) receptors on astrocytes is questioned.

  13. Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases*

    PubMed Central

    Sacco, Francesca; Tinti, Michele; Palma, Anita; Ferrari, Emanuela; Nardozza, Aurelio P.; van Huijsduijnen, Rob Hooft; Takahashi, Takamune; Castagnoli, Luisa; Cesareni, Gianni

    2009-01-01

    Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition. PMID:19494114

  14. Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2.

    PubMed

    Lee, Yun Yeong; Ryu, Min Sook; Kim, Hong Seok; Suganuma, Masami; Song, Kye Yong; Lim, In Kyoung

    2016-03-01

    The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS(104) via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21(WAF1) gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.

  15. Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2.

    PubMed

    Lee, Yun Yeong; Ryu, Min Sook; Kim, Hong Seok; Suganuma, Masami; Song, Kye Yong; Lim, In Kyoung

    2016-03-01

    The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS(104) via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21(WAF1) gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells. PMID:26912086

  16. Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2

    PubMed Central

    Lee, Yun Yeong; Ryu, Min Sook; Kim, Hong Seok; Suganuma, Masami; Song, Kye Yong; Lim, In Kyoung

    2016-01-01

    The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS104 via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21WAF1 gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells. PMID:26912086

  17. Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells

    PubMed Central

    2013-01-01

    Background The naphthoquinone pigment, shikonin, is a major component of Lithospermum erythrorhizon and has been shown to have various biological functions, including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we investigated the effect of shikonin on adipocyte differentiation and its mechanism of action in 3T3-L1 cells. Methods To investigate the effects of shikonin on adipocyte differentiation, 3T3-L1 cells were induced to differentiate using 3-isobutyl-1-methylzanthine, dexamethasone, and insulin (MDI) for 8 days in the presence of 0–2 μM shikonin. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the anti-adipogenic mechanism of shikonin, adipogenic transcription factors, the phosphorylation levels of ERK, and adipogenic gene expression were analyzed by Western blotting and quantitative real-time PCR. To further confirm that shikonin inhibits adipogenic differentiation through downregulation of ERK 1/2 activity, 3T3-L1 cells were treated with shikonin in the presence of FGF-2, an activator, or PD98059, an inhibitor, of the ERK1/2 signaling pathway. Results Shikonin effectively suppressed adipogenesis and downregulated the protein levels of 2 major transcription factors, PPARγ and C/EBPα, as well as the adipocyte specific gene aP2 in a dose-dependent manner. qRT-PCR analysis revealed that shikonin inhibited mRNA expression of adipogenesis-related genes, such as PPARγ, C/EBPα, and aP2. Adipocyte differentiation was mediated by ERK 1/2 phosphorylation, which was confirmed by pretreatment with PD98059 (an ERK 1/2 inhibitor) or FGF-2 (an ERK 1/2 activator). The phosphorylation of ERK1/2 during the early stages of adipogenesis in 3T3-L1 cells was inhibited by shikonin. We also confirmed that FGF-2-stimulated ERK 1/2 activity was attenuated by shikonin. Conclusions These results demonstrate that shikonin inhibits adipogenic differentiation via suppression of the ERK signaling pathway

  18. ACTIVATION OF EXTRACELLULAR-SIGNAL REGULATED KINASE (ERK1/2) BY FLUID SHEAR IS CA2+- AND ATP-DEPENDENT IN MC3T3-E1 OSTEOBLASTS

    PubMed Central

    Liu, Dawei; Genetos, Damian C.; Shao, Ying; Geist, Derik J.; Li, Jiliang; Ke, Hua Zhu; Turner, Charles H.; Duncan, Randall L.

    2010-01-01

    To determine the role of Ca2+ signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca2+ signaling during application of fluid shear stress (FSS). FSS only activated ERK1/2, rapidly inducing phosphorylation within 5 minutes of the onset of shear. Phosphorylation of ERK1/2 (pERK1/2) was significantly reduced when Ca2+i was chelated with BAPTA or when Ca2+ was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca2+ channel and the mechanosensitive cation-selective channel blocked FSS-induced pERK1/2. Inhibition of phospholipase C with U73122 significantly reduced pERK1/2. This inhibition did not result from block of intracellular Ca2+ release, but a loss of PKC activation. Recent data suggests a role of ATP release and purinergic receptor activation in mechanotransduction. Apyrase-mediated hydrolysis of extracellular ATP completely blocked FSS-induced phosphorylation of ERK1/2, while addition of exogenous ATP to static cells mimicked the effects of FSS on pERK1/2. Two P2 receptors, P2Y2 and P2X7, have been associated with the anabolic responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X7 knockout mice, we found that the P2X7, but not the P2Y2, purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca2+-dependent ATP release, however both increased Ca2+i and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X7, but not P2Y2, purinergic receptors. PMID:18291742

  19. CacyBP/SIP binds ERK1/2 and affects transcriptional activity of Elk-1

    SciTech Connect

    Kilanczyk, Ewa; Filipek, Slawomir; Jastrzebska, Beata; Filipek, Anna

    2009-02-27

    In this work we showed for the first time that mouse CacyBP/SIP interacts with extracellular signal regulated kinases 1 and 2 (ERK1/2). We also established that a calcium binding protein, S100A6, competes for this interaction. Moreover, the E217K mutant of CacyBP/SIP does not bind significantly to ERK1/2 although it retains the ability to interact with S100A6. Molecular modeling shows that the E217K mutation in the 189-219 CacyBP/SIP fragment markedly changes its electrostatic potential, suggesting that the binding with ERK1/2 might have an electrostatic character. We also demonstrate that CacyBP/SIP-ERK1/2 interaction inhibits phosphorylation of the Elk-1 transcription factor in vitro and in the nuclear fraction of NB2a cells. Altogether, our data suggest that the binding of CacyBP/SIP with ERK1/2 might regulate Elk-1 phosphorylation/transcriptional activity and that S100A6 might further modulate this effect via Ca{sup 2+}-dependent interaction with CacyBP/SIP and competition with ERK1/2.

  20. Astrocyte elevated gene-1 regulates CCL3/CCR5-induced epithelial-to-mesenchymal transition via Erk1/2 and Akt signaling in cardiac myxoma.

    PubMed

    Shi, Ping; Fang, Changcun; Pang, Xinyan

    2015-09-01

    In recent years, astrocyte elevated gene-1 (AEG-1) has been reported as a key mediator that is involved in the epithelial-to-mesenchymal transition (EMT) process. However, the mechanisms underlying CCL3/CCR5-AEG-1 pathway-mediated EMT in cardiac myxoma (CM) has not been well featured till now. We used immnohistochemistry and immunoblotting to assess the expression of CCR5 and AEG-1 in 30 cases of CM tissues and cells. Subsequently, cultured CM cells were treated with si-AEG-1 or si-CCR5 and then subjected to in vitro assays. We observed that CCR5 and AEG-1 proteins were highly expressed in CM tissues (73.3 and 76.7%, respectively) and closely correlated with tumor size (>5 cm). Importantly, we validated the expression of AEG-1, p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 increased in the CM cell with CCL3 treatment in a time- and concentration-dependent manner. When CM cells were treated with si-CCR5, the expression of AEG-1, p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 was downregulated. In addition, when CM cells were treated with si-AEG-1, the expression of p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 was also downregulated. Using the cell cycle and proliferation assay, the knockdown of AEG-1 inhibited the entry of G1 into S phase and the proliferation capacity of CM cells. In conclusion, AEG-1 mediates CCL3/CCR5-induced EMT development via both Erk1/2 and Akt signaling pathway in CM patients, which indicates CCL3/CCR5-AEG-1-EMT pathway could be suggested as a useful target to affect the progression of CM.

  1. Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

    PubMed

    Rodríguez-Peña, Ana B; Grande, Maria T; Eleno, Nélida; Arévalo, Miguel; Guerrero, Carmen; Santos, Eugerio; López-Novoa, José M

    2008-07-01

    Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

  2. Angiotensin II increases matrix metalloproteinase 2 expression in human aortic smooth muscle cells via AT1R and ERK1/2

    PubMed Central

    Wang, Chunmao; Qian, Xiangyang; Sun, Xiaogang

    2015-01-01

    Increased levels of angiotensin II (Ang II) and activated matrix metalloproteinase 2 (MMP-2) produced by human aortic smooth muscle cells (human ASMCs) have recently been implicated in the pathogenesis of thoracic aortic aneurysm (TAA). Additionally, angiotensin II type 1 receptor (AT1R)-mediated extracellular signal-regulated kinase (ERK)1/2 activation contributes to TAA development in Marfan Syndrome. However, there is scant data regarding the relationship between Ang II and MMP-2 expression in human ASMCs. Therefore, we investigated the effect of Ang II on MMP-2 expression in human ASMCs and used Western blotting to identify the Ang II receptors and intracellular signaling pathways involved. Reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence data demonstrated that Ang II receptors were expressed on human ASMCs. Additionally, Ang II increased the expression of Ang II type 2 receptor (AT2R) but not AT1R at both the transcriptional and translational levels. Furthermore, Western blotting showed that Ang II increased MMP-2 expression in human ASMCs in a dose- and time-dependent manner. This response was completely inhibited by the AT1R inhibitor candesartan but not by the AT2R blocker PD123319. In addition, Ang II–induced upregulation of MMP-2 was mediated by the activation of ERK1/2, whereas p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) had no effect on this process. In conclusion, these results indicate that Ang II can increase the expression of MMP-2 via AT1 receptor and ERK1/2 signaling pathways in human ASMCs and suggest that antagonists of AT1R and ERK1/2 may be useful for treating TAAs. PMID:25767191

  3. Length of stay an important mediator of hospital-acquired methicillin-resistant Staphylococcus aureus.

    PubMed

    Wong, J G; Chen, M I; Win, M K; Ng, P Y; Chow, A

    2016-04-01

    Hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) is becoming increasingly established in Asian hospitals. The primary aim of this study was to decompose the risk factors for HA-MRSA based on conceptual clinical pathways. The secondary aim was to show the amount of effect attributable to antibiotic exposure and total length of stay before outcome (LBO) so that institutions can manage at-risk patients accordingly. A case-control study consisting of 1200 inpatients was conducted in a large tertiary hospital in Singapore between January and December 2006. Results from the generalized structural equation model (GSEM) show that LBO [adjusted odds ratio (aOR) 14·9, 95% confidence interval (CI) 8·7-25·5], prior hospitalization (aOR 6·2, 95% CI 3·3-11·5), and cumulative antibiotic exposure (aOR 3·5, 95% CI 2·3-5·3), directly affected HA-MRSA acquisition. LBO accounted for the majority of the effects due to age (100%), immunosuppression (67%), and surgery (96%), and to a lesser extent for male gender (22%). Our model enabled us to account and quantify effects of intermediaries. LBO was found to be an important mediator of age, immunosuppression and surgery on MRSA infection. Traditional regression approaches will not only give different conclusions but also underestimate the effects. Hospitals should minimize the hospital stay when possible to reduce the risk of MRSA.

  4. Fibrocyte-like cells mediate acquired resistance to anti-angiogenic therapy with bevacizumab

    PubMed Central

    Mitsuhashi, Atsushi; Goto, Hisatsugu; Saijo, Atsuro; Trung, Van The; Aono, Yoshinori; Ogino, Hirokazu; Kuramoto, Takuya; Tabata, Sho; Uehara, Hisanori; Izumi, Keisuke; Yoshida, Mitsuteru; Kobayashi, Hiroaki; Takahashi, Hidefusa; Gotoh, Masashi; Kakiuchi, Soji; Hanibuchi, Masaki; Yano, Seiji; Yokomise, Hiroyasu; Sakiyama, Shoji; Nishioka, Yasuhiko

    2015-01-01

    Bevacizumab exerts anti-angiogenic effects in cancer patients by inhibiting vascular endothelial growth factor (VEGF). However, its use is still limited due to the development of resistance to the treatment. Such resistance can be regulated by various factors, although the underlying mechanisms remain incompletely understood. Here we show that bone marrow-derived fibrocyte-like cells, defined as alpha-1 type I collagen-positive and CXCR4-positive cells, contribute to the acquired resistance to bevacizumab. In mouse models of malignant pleural mesothelioma and lung cancer, fibrocyte-like cells mediate the resistance to bevacizumab as the main producer of fibroblast growth factor 2. In clinical specimens of lung cancer, the number of fibrocyte-like cells is significantly increased in bevacizumab-treated tumours, and correlates with the number of treatment cycles, as well as CD31-positive vessels. Our results identify fibrocyte-like cells as a promising cell biomarker and a potential therapeutic target to overcome resistance to anti-VEGF therapy. PMID:26635184

  5. Phasic phosphorylation of caldesmon and ERK 1/2 during contractions in human myometrium.

    PubMed

    Paul, Jonathan; Maiti, Kaushik; Read, Mark; Hure, Alexis; Smith, Julia; Chan, Eng-Cheng; Smith, Roger

    2011-01-01

    Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.

  6. Cross talk between MMP2-Spm-Cer-S1P and ERK1/2 in proliferation of pulmonary artery smooth muscle cells under angiotensin II stimulation.

    PubMed

    Chowdhury, Animesh; Sarkar, Jaganmay; Pramanik, Pijush Kanti; Chakraborti, Tapati; Chakraborti, Sajal

    2016-08-01

    The aim of the present study is to establish the mechanism associated with the proliferation of PASMCs under ANG II stimulation. The results showed that treatment of PASMCs with ANG II induces an increase in cell proliferation and 100 nM was the optimum concentration for maximum increase in proliferation of the cells. Pretreatment of the cells with AT1, but not AT2, receptor antagonist inhibited ANG II induced cell proliferation. Pretreatment with pharmacological and genetic inhibitors of sphingomyelinase (SMase) and sphingosine kinase (SPHK) prevented ANG II-induced cell proliferation. ANG II has also been shown to induce SMase activity, SPHK phosphorylation and S1P production. In addition, ANG II caused an increase in proMMP-2 expression and activation, ERK1/2 phosphorylation and NADPH oxidase activation. Upon inhibition of MMP-2, SMase activity and S1P level were curbed leading to inhibition of cell proliferation. SPHK was phosphorylated by ERK1/2 during ET-1 stimulation of the cells. ANG II-induced ERK1/2 phosphorylation and proMMP-2 expression and activation in the cells were abrogated upon inhibition of NADPH oxidase activity. Overall, NADPH oxidase plays an important role in proMMP-2 expression and activation and that MMP-2 mediated SMC proliferation occurs through the involvement of Spm-Cer-S1P signaling axis under ANG II stimulation of PASMCs. PMID:27210740

  7. Hypoxia activates 15-PGDH and its metabolite 15-KETE to promote pulmonary artery endothelial cells proliferation via ERK1/2 signalling

    PubMed Central

    Ma, Cui; Liu, Yun; Wang, Yanyan; Zhang, Chen; Yao, Hongmin; Ma, Jun; Zhang, Lei; Zhang, Dandan; Shen, Tingting; Zhu, Daling

    2014-01-01

    BACKGROUND AND PURPOSE Dysfunction and injury of endothelial cells in the pulmonary artery play critical roles in the hypertension induced by chronic hypoxia. One consequence of hypoxia is increased activity of 15-hydroxyprostaglandin dehydrogenase (PGDH). Here, we have explored, in detail, the effects of hypoxia on the proliferation of pulmonary artery endothelial cells. EXPERIMENTAL APPROACH We used bromodeoxyuridine incorporation, cell-cycle analysis, immunohistochemistry and Western blot analysis to study the effects of hypoxia, induced 15-PGDH) activity and its product, 15-keto-6Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15-KETE), on endothelial cell proliferation. Scratch-wound and tube formation assays were also used to study migration of endothelial cells. KEY RESULTS 15-KETE increased DNA synthesis and enhanced the transition from the G0/G1 phase to the S phase in hypoxia. Inhibition of 15-PGDH or siRNA for 15-PGDH reversed these effects. 15-KETE also activated the ERK1/2 signalling pathway. 15-KETE-induced cell migration and tube formation were reversed by blocking ERK1/2, but not the p38 MAPK pathway. CONCLUSIONS AND IMPLICATIONS Hypoxia-induced endothelial proliferation and migration, an important underlying mechanism contributing to hypoxic pulmonary vascular remodelling, appears to be mediated by 15-PGDH and 15-KETE, via the ERK1/2 signalling pathway. PMID:24467360

  8. Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ape1/Ref-1) is essential for IL-21-induced signal transduction through ERK1/2 pathway

    SciTech Connect

    Juliana, Farha M.; Nara, Hidetoshi; Onoda, Tadashi; Rahman, Mizanur; Araki, Akemi; Jin, Lianjin; Fujii, Hodaka; Tanaka, Nobuyuki; Hoshino, Tomoaki; Asao, Hironobu

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer IL-21 induces nuclear accumulation of Ape1/Ref-1 protein. Black-Right-Pointing-Pointer Ape1/Ref-1 is indispensable in IL-21-induced cell proliferation and survival signal. Black-Right-Pointing-Pointer Ape1/Ref-1 is required for IL-21-induced ERK1/2 activation. -- Abstract: IL-21 is a pleiotropic cytokine that regulates T-cell and B-cell differentiation, NK-cell activation, and dendritic cell functions. IL-21 activates the JAK-STAT, ERK, and PI3K pathways. We report here that Ape1/Ref-1 has an essential role in IL-21-induced cell growth signal transduction. Overexpression of Ape1/Ref-1 enhances IL-21-induced cell proliferation, but it is suppressed by overexpressing an N-terminal deletion mutant of Ape1/Ref-1 that lacks the redox domain. Furthermore, knockdown of the Ape1/Ref-1 mRNA dramatically compromises IL-21-induced ERK1/2 activation and cell proliferation with increasing cell death. These impaired activities are recovered by the re-expression of Ape1/Ref-1 in the knockdown cells. Our findings are the first demonstration that Ape1/Ref-1 is an indispensable molecule for the IL-21-mediated signal transduction through ERK1/2 activation.

  9. Kruppel-like factor 2 inhibit the angiogenesis of cultured human liver sinusoidal endothelial cells through the ERK1/2 signaling pathway.

    PubMed

    Zeng, Xiao-Qing; Li, Na; Pan, Du-Yi; Miao, Qing; Ma, Gui-Fen; Liu, Yi-Mei; Tseng, Yu-Jen; Li, Feng; Xu, Li-Li; Chen, Shi-Yao

    2015-09-01

    Kruppel-like factor 2 (KLF2) is a crucial anti-angiogenic factor. However, its precise role in hepatic angiogenesis induced by liver sinusoidal endothelial cells (LSECs) remain unclear. This study was aimed to evaluate the effect of KLF2 on angiogenesis of LSECs and to explore the corresponding mechanism. Cultured human LSECs were infected with different lentiviruses to overexpress or suppress KLF2 expression. The CCK-8 assay, transwell migration assay and tube formation test, were used to investigate the roles of KLF2 in the proliferation, migration and vessel tube formation of LSECs, respectively. The expression and phosphorylation of ERK1/2 were detected by western blot. We discovered that the up-regulation of KLF2 expression dramatically inhibited proliferation, migration and tube formation in treated LSECs. Correspondingly, down-regulation of KLF2 expression significantly promoted proliferation, migration and tube formation in treated LSECs. Additionally, KLF2 inhibited the phosphorylation of ERK1/2 pathway, followed by the function of KLF2 in the angiogenesis of LSECs disrupted. In conclusion, KLF2 suppressed the angiogenesis of LSECs through inhibition of cell proliferation, migration, and vessel tube formation. These functions of KLF2 may be mediated through the ERK1/2 signaling pathway.

  10. Integrin αv promotes proliferation by activating ERK 1/2 in the human lung cancer cell line A549.

    PubMed

    Fu, Shijie; Fan, Limin; Pan, Xufeng; Sun, Yifeng; Zhao, Heng

    2015-02-01

    Lung cancer is a leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) constitutes ~85% of lung cancers. However, the mechanisms underlying the progression of NSCLC remain unclear. In this study, we found the mRNA and protein expression levels of integrin αv are both increased in NSCLC tissues compared to healthy ones, which indicates that integrin αv may play an important role in NSCLC progression. To further investigate the roles of integrin αv in NSCLC, we overexpressed the integrin αv gene in the NSCLC cell line A549, and found that the cell proliferative ability increased. The apoptosis of A549 cells was inhibited with overexpression of integrin αv. To elucidate the molecular mechanism underlying the role of integrin αv in promoting NSCLC progression, we studied the expression of proteins from a number of important pathways associated with tumorigenesis, and found that the extracellular signal regulated protein kinase (ERK)1/2 signaling pathway may be involved in the mediation of the observed integrin αv effects. component of an important pathway for tumorigenesis, the ERK 1/2. Following inhibition of ERK 1/2 signaling, the proliferation of A549 cells induced by integrin αv was reduced, while the inhibition of apoptosis was attenuated. Our findings demonstrate that integrin αv promotes the proliferation of the human lung cancer cell line A549 by activating the ERK 1/2 signaling pathway, which suggests that this pathway may be a promising target for the treatment of human lung cancer.

  11. Ochratoxin A activates opposing c-MET/PI3K/Akt and MAPK/ERK 1-2 pathways in human proximal tubule HK-2 cells.

    PubMed

    Özcan, Zeynep; Gül, Gizem; Yaman, Ibrahim

    2015-08-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by filamentous fungi, such as Aspergillus and Penicillium. Because OTA is a common contaminant of food and feeds, humans and animals are frequently exposed to OTA in daily life. It has been classified as a carcinogen in rodents and a possible carcinogen in humans. OTA has been shown to deregulate a variety of different signal transduction pathways in a cell type- and dosage-depending manner resulting in contrasting physiological effects, such as survival or cell death. While the ERK1-2 and JNK/SAPK MAPK pathways are major targets, knowledge about their role in OTA-mediated cell survival and death is fragmented. Similarly, the contribution of the PI3K/Akt pathway to the carcinogenic effect of OTA in proximal tubule cells has not been elucidated in detail. In this study, we demonstrated that OTA induced sustained activation of the PI3K/Akt and MEK/ERK1-2 signaling pathways in a dose- and time-dependent manner in HK-2 cells. Chemical inhibition of ERK1-2 activation or overexpression of dominant-negative and kinase-dead MEK1 leads to increased cell viability and decreased apoptosis in OTA-treated cells. Blockage of PI3K/Akt with Wortmannin aggravated the negative effect of OTA on cell viability and increased the levels of apoptosis. Moreover, we identified the c-MET proto-oncogene as an upstream receptor tyrosine kinase responsible for OTA-induced activation of PI3K/Akt signaling in HK-2 cells. Our data suggest that OTA may potentiate carcinogenesis by sustained activation of c-MET/PI3K/Akt signaling through suppression of apoptosis induced by MEK/ERK1-2 activation in damaged renal proximal tubule epithelial cells. PMID:25002221

  12. Erianin inhibits high glucose-induced retinal angiogenesis via blocking ERK1/2-regulated HIF-1α-VEGF/VEGFR2 signaling pathway

    PubMed Central

    Yu, Zengyang; Zhang, Tianyu; Gong, Chenyuan; Sheng, Yuchen; Lu, Bin; Zhou, Lingyu; Ji, Lili; Wang, Zhengtao

    2016-01-01

    Erianin is a natural compound found in Dendrobium chrysotoxum Lindl. Diabetic retinopathy (DR) is a serious and common microvascular complication of diabetes. This study aims to investigate the inhibitory mechanism of erianin on retinal neoangiogenesis and its contribution to the amelioration of DR. Erianin blocked high glucose (HG)-induced tube formation and migration in choroid-retinal endothelial RF/6A cells. Erianin inhibited HG-induced vascular endothelial growth factor (VEGF) expression, hypoxia-inducible factor 1-alpha (HIF-1α) translocation into nucleus and ERK1/2 activation in RF/6A and microglia BV-2 cells. MEK1/2 inhibitor U0126 blocked HG-induced HIF-1α and ERK1/2 activation in both above two cells. In addition, erianin abrogated VEGF-induced angiogenesis in vitro and in vivo, and also inhibited VEGF-induced activation of VEGF receptor 2 (VEGFR2) and its downstream cRaf-MEK1/2-ERK1/2 and PI3K-AKT signaling pathways in RF/6A cells. Furthermore, erianin reduced the increased retinal vessels, VEGF expression and microglia activation in streptozotocin (STZ)-induced hyperglycemic and oxygen-induced retinopathy (OIR) mice. In conclusion, our results demonstrate that erianin inhibits retinal neoangiogenesis by abrogating HG-induced VEGF expression by blocking ERK1/2-mediated HIF-1α activation in retinal endothelial and microglial cells, and further suppressing VEGF-induced activation of VEGFR2 and its downstream signals in retinal endothelial cells. PMID:27678303

  13. Differential regulation of proliferation and neuronal differentiation in adult rat spinal cord neural stem/progenitors by ERK1/2, Akt, and PLCγ

    PubMed Central

    Chan, Wai Si; Sideris, Alexandra; Sutachan, Jhon J.; Montoya G, Jose V.; Blanck, Thomas J. J.; Recio-Pinto, Esperanza

    2013-01-01

    Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs. PMID:23986655

  14. An NF-κB-independent and Erk1/2-dependent mechanism controls CXCL8/IL-8 responses of airway epithelial cells to cadmium.

    PubMed

    Cormet-Boyaka, Estelle; Jolivette, Kalyn; Bonnegarde-Bernard, Astrid; Rennolds, Jessica; Hassan, Fatemat; Mehta, Payal; Tridandapani, Susheela; Webster-Marketon, Jeanette; Boyaka, Prosper N

    2012-02-01

    Airway epithelial cells in the lung are the first line of defense against pathogens and environmental pollutants. Inhalation of the environmental pollutant cadmium has been linked to the development of lung cancer and chronic obstructive pulmonary disease, which are diseases characterized by chronic inflammation. To address the role of airway epithelial cells in cadmium-induced lung inflammation, we investigated how cadmium regulates secretion of interleukin 8 (IL-8) by airway epithelial cells. We show that exposure of human airway epithelial cells to subtoxic doses of cadmium in vitro promotes a characteristic inflammatory cytokine response consisting of IL-8, but not IL-1β or tumor necrosis factor-alpha. We also found that intranasal delivery of cadmium increases lung levels of the murine IL-8 homologs macrophage inflammatory protein-2 and keracinocyte-derived chemokine and results in an influx of Gr1+ cells into the lung. We determined that inhibition of the nuclear factor-κB (NF-κB) pathway had no effect on cadmium-induced IL-8 secretion by human airway epithelial cells, suggesting that IL-8 production was mediated through an NF-κB-independent pathway. Mitogen-activated protein kinases (MAPKs) are often involved in proinflammatory signaling. Cadmium could activate the main MAPKs (i.e., p38, JNK, and Erk1/2) in human airway epithelial cells. However, only pharmacological inhibition of Erk1/2 pathway or knockdown of the expression of Erk1 and Erk2 using small interfering RNAs suppressed secretion of IL-8 induced by cadmium. Our findings identify cadmium as a potent activator of the proinflammatory cytokine IL-8 in lung epithelial cells and reveal for the first time the role of an NF-κB-independent but Erk1/2-dependent pathway in cadmium-induced lung inflammation. PMID:22094458

  15. Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia

    PubMed Central

    Hellyer, Thomas P; Conway Morris, Andrew; McAuley, Daniel F; Walsh, Timothy S; Anderson, Niall H; Singh, Suveer; Dark, Paul; Roy, Alistair I; Baudouin, Simon V; Wright, Stephen E; Perkins, Gavin D; Kefala, Kallirroi; Jeffels, Melinda; McMullan, Ronan; O'Kane, Cecilia M; Spencer, Craig; Laha, Shondipon; Robin, Nicole; Gossain, Savita; Gould, Kate; Ruchaud-Sparagano, Marie-Hélène; Scott, Jonathan; Browne, Emma M; MacFarlane, James G; Wiscombe, Sarah; Widdrington, John D; Dimmick, Ian; Laurenson, Ian F; Nauwelaers, Frans; Simpson, A John

    2015-01-01

    Background Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. Objectives We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. Methods A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >104 colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. Results Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). Conclusions Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship. PMID:25298325

  16. MAP KINASE ERK 1/2 INHIBITORS INDUCE DYSMORPHOLOGY IN MOUSE WHOLE EMBRYO CULTURE

    EPA Science Inventory

    ROSEN, M.B. and E. S. HUNTER. Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina. MAP kinase Erk1/2 inhibitors induce dysmorphology in mouse whole embryo culture.

    MAP Kinase signal transduction is associated with a variety ...

  17. Serotonin increases ERK1/2 phosphorylation in astrocytes by stimulation of 5-HT2B and 5-HT2C receptors.

    PubMed

    Li, Baoman; Zhang, Shiquen; Li, Min; Hertz, Leif; Peng, Liang

    2010-11-01

    We have previously shown that fluoxetine causes ERK(1/2) phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT(2B) receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK(1/2) phosphorylation by stimulation of 5-HT(2B) receptors with high affinity (EC(50): 20-30 pM), and by stimulation of 5-HT(2C) receptor with low affinity (EC(50): 1 microM or higher). ERK(1/2) phosphorylation induced by stimulation of either 5-HT(2B) or 5-HT(2C) receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT(2B) receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT(2B)-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT(2B) receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT(2B) receptors. In contrast the relevance of the observed 5-HT(2C) receptors on astrocytes is questioned. PMID:20450948

  18. Grifolin directly targets ERK1/2 to epigenetically suppress cancer cell metastasis.

    PubMed

    Luo, Xiangjian; Yang, Lifang; Xiao, Lanbo; Xia, Xiaofeng; Dong, Xin; Zhong, Juanfang; Liu, Ying; Li, Namei; Chen, Ling; Li, Hongde; Li, Wei; Liu, Wenbin; Yu, Xinfang; Chen, Hanyong; Tang, Min; Weng, Xinxian; Yi, Wei; Bode, Ann; Dong, Zigang; Liu, Jikai; Cao, Ya

    2015-12-15

    Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been reported by us and others to display potent antitumor effects. However, the molecular target of grifolin has not been identified and the underlying mechanism of action is not fully understood. Here, we report that the ERK1/2 protein kinases are direct molecular targets of grifolin. Molecular modeling, affinity chromatography and fluorescence quenching analyses showed that grifolin directly binds to ERK1/2. And in vitro and ex vivo kinase assay data further demonstrated that grifolin inhibited the kinase activities of ERK1/2. We found that grifolin suppressed adhesion, migration and invasion of high-metastatic cancer cells. The inhibitory effect of grifolin against tumor metastasis was further confirmed in a metastatic mouse model. We found that grifolin decreased phosphorylation of Elk1 at Ser383, and the protein as well as the mRNA level of DNMT1 was also down-regulated. By luciferase reporter and ChIP assay analyses, we confirmed that grifolin inhibited the transcription activity of Elk1 as well as its binding to the dnmt1 promoter region. Moreover, we report that significant increases in the mRNA levels of Timp2 and pten were induced by grifolin. Thus, our data suggest that grifolin exerts its anti-tumor activity by epigenetic reactivation of metastasis inhibitory-related genes through ERK1/2-Elk1-DNMT1 signaling. Grifolin may represent a promising therapeutic lead compound for intervention of cancer metastasis, and it may also be useful as an ERK1/2 kinase inhibitor as well as an epigenetic agent to further our understanding of DNMT1 function.

  19. Grifolin directly targets ERK1/2 to epigenetically suppress cancer cell metastasis

    PubMed Central

    Xiao, Lanbo; Xia, Xiaofeng; Dong, Xin; Zhong, Juanfang; Liu, Ying; Li, Namei; Chen, Ling; Li, Hongde; Li, Wei; Liu, Wenbin; Yu, Xinfang; Chen, Hanyong; Tang, Min; Weng, Xinxian; Yi, Wei; Bode, Ann; Dong, Zigang; Liu, Jikai; Cao, Ya

    2015-01-01

    Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been reported by us and others to display potent antitumor effects. However, the molecular target of grifolin has not been identified and the underlying mechanism of action is not fully understood. Here, we report that the ERK1/2 protein kinases are direct molecular targets of grifolin. Molecular modeling, affinity chromatography and fluorescence quenching analyses showed that grifolin directly binds to ERK1/2. And in vitro and ex vivo kinase assay data further demonstrated that grifolin inhibited the kinase activities of ERK1/2. We found that grifolin suppressed adhesion, migration and invasion of high-metastatic cancer cells. The inhibitory effect of grifolin against tumor metastasis was further confirmed in a metastatic mouse model. We found that grifolin decreased phosphorylation of Elk1 at Ser383, and the protein as well as the mRNA level of DNMT1 was also down-regulated. By luciferase reporter and ChIP assay analyses, we confirmed that grifolin inhibited the transcription activity of Elk1 as well as its binding to the dnmt1 promoter region. Moreover, we report that significant increases in the mRNA levels of Timp2 and pten were induced by grifolin. Thus, our data suggest that grifolin exerts its anti-tumor activity by epigenetic reactivation of metastasis inhibitory-related genes through ERK1/2-Elk1-DNMT1 signaling. Grifolin may represent a promising therapeutic lead compound for intervention of cancer metastasis, and it may also be useful as an ERK1/2 kinase inhibitor as well as an epigenetic agent to further our understanding of DNMT1 function. PMID:26516701

  20. ERK1 and ERK2 Map Kinases: Specific Roles or Functional Redundancy?

    PubMed Central

    Buscà, Roser; Pouysségur, Jacques; Lenormand, Philippe

    2016-01-01

    The MAP kinase signaling cascade Ras/Raf/MEK/ERK has been involved in a large variety of cellular and physiological processes that are crucial for life. Many pathological situations have been associated to this pathway. More than one isoform has been described at each level of the cascade. In this review we devoted our attention to ERK1 and ERK2, which are the effector kinases of the pathway. Whether ERK1 and ERK2 specify functional differences or are in contrast functionally redundant, constitutes an ongoing debate despite the huge amount of studies performed to date. In this review we compiled data on ERK1 vs. ERK2 gene structures, protein sequences, expression levels, structural and molecular mechanisms of activation and substrate recognition. We have also attempted to perform a rigorous analysis of studies regarding the individual roles of ERK1 and ERK2 by the means of morpholinos, siRNA, and shRNA silencing as well as gene disruption or gene replacement in mice. Finally, we comment on a recent study of gene and protein evolution of ERK isoforms as a distinct approach to address the same question. Our review permits the evaluation of the relevance of published studies in the field especially when measurements of global ERK activation are taken into account. Our analysis favors the hypothesis of ERK1 and ERK2 exhibiting functional redundancy and points to the concept of the global ERK quantity, and not isoform specificity, as being the essential determinant to achieve ERK function. PMID:27376062

  1. The de-ubiquitinase UCHL1 promotes gastric cancer metastasis via the Akt and Erk1/2 pathways.

    PubMed

    Gu, Yu-yu; Yang, Mei; Zhao, Mei; Luo, Qing; Yang, Lin; Peng, Hua; Wang, Jia; Huang, Sheng-kai; Zheng, Zhao-xu; Yuan, Xing-hua; Liu, Ping; Huang, Chang-zhi

    2015-11-01

    Ubiquitin C-terminal hydrolase-L1 (UCHL1) is a de-ubiquitinating enzyme, which enzymatic activity relies on the C90 site. The function of UCHL1 is controversial in different types of cancer, and its role in gastric cancer progression remains unclear. In this study, immunohistochemistry staining was applied to detect the expression of UCHL1 in primary gastric cancer and liver metastases from gastric cancer. MKN45 and BGC823 cell lines with stable expression of de-ubiquitinase active UCHL1 or inactive UCHL1-variant C90S were established by lentiviral infection. The effect of UCHL1 on cell proliferation was evaluated by MTT and colony formation assays. The abilities of cell migration and invasion were determined by transwell assay. Protein expression levels were determined by Western blot. The results indicated that UCHL1 had a significantly higher positive expression rate in liver metastases from gastric cancer compared with primary gastric cancer. Overexpression of UCHL1 in MKN45 and BGC823 cells promoted cell proliferation, migration, and invasion depending on its de-ubiquitinase activity. UCHL1 activated Akt and Erk1/2, which process also required enzymatic activity and was necessary for mediating cell migration and invasion. These findings demonstrated that UCHL1 promoted cell proliferation, migration, and invasion depending on its de-ubiquitinase activity by activating Akt and Erk1/2, which may account for its higher positive expression rate in liver metastases from gastric cancer. UCHL1 could be a candidate biomarker and a therapeutic target for gastric cancer metastasis. PMID:26018507

  2. The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

    SciTech Connect

    Wang, Ji-meng; Zhao, Hong-xi; Wang, Li; Gao, Zhi-ying; Yao, Yuan-qing

    2013-05-10

    Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

  3. Overexpression of cyclooxygenase-2 in NCI-H292 human alveolar epithelial carcinoma cells: roles of p38 MAPK, ERK-1/2, and PI3K/PKB signaling proteins.

    PubMed

    Sung, Suhaeng; Park, Yukyoung; Jo, Jeong-Rang; Jung, Nak-Kyun; Song, Dae-Kyu; Bae, Jaehoon; Keum, Dong-Yun; Kim, Jae-Bum; Park, Gy-Young; Jang, Byeong-Churl; Park, Jong-Wook

    2011-10-01

    Evidence suggests overexpression of COX-2 and its role in many human cancers, including lung. However, the regulatory mechanism underlying COX-2 overexpression in lung cancer is not fully understood. We herein investigated whether COX-2 is overexpressed in human airway cancer cell lines, including A549 (lung), Hep-2 (bronchial), and NCI-H292 (alveolar). When grown in cell culture medium containing 10% FBS (serum), of note, there was strong and transient induction of COX-2 protein and mRNA in NCI-H292 cells, but little or low COX-2 expression is seen in A549 or Hep-2 cells. Interestingly, strong and sustained activities of ERK-1/2, JNK-1/2, p38 MAPK, and PKB were also shown in NCI-H292 cells grown in presence of serum. Profoundly, results of pharmacological inhibition studies demonstrated that the serum-dependent COX-2 up-regulation in NCI-H292 cells is attributed to not only the p38 MAPK-, PI3K/PKB-, and ERK-1/2-mediated COX-2 transcriptional up-regulation but also the p38 MAPK- and ERK-1/2-mediated post-transcriptional COX-2 mRNA stabilization. Of further note, it was shown that the ERK-1/2 and PI3K/PKB (but not COX-2, p38 MAPK, and JNK-1/2) activities are necessary for growth of NCI-H292 cells. These findings collectively demonstrate for the first time that COX-2 expression is transiently up-regulated by serum addition in NCI-H292 cells and the serum-induced COX-2 expression is closely linked to the p38 MAPK-, ERK-1/2-, and PI3K/PKB-mediated COX-2 transcriptional and post-transcriptional up-regulation.

  4. Polydatin induces bone marrow stromal cells migration by activation of ERK1/2.

    PubMed

    Chen, ZhenQiu; Wei, QiuShi; Hong, GuoJu; Chen, Da; Liang, Jiang; He, Wei; Chen, Mei Hui

    2016-08-01

    Bone marrow stromal cells (BMSCs) have proven to be useful for the treatment of numerous human diseases. However, the reparative ability of BMSCs is limited by their poor migration. Polydatin, widely used in traditional Chinese remedies, has proven to exert protective effects to BMSCs. However, little is known about its role in BMSCs migration. In this study, we studied the effects of polydatin on rat BMSCs migration using the scratch wound healing and transwell migration assays. Our results showed polydatin could promote BMSCs migration. Further experiments showed activation of ERK 1/2, but not JNK, was required for polydatin-induced BMSCs migration, suggesting that polydatin may promote BMSCs migration via the ERK 1/2 signaling pathways. Taken together, our results indicate that polydatin might be beneficial for stem cell replacement therapy by improving BMSCs migration.

  5. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  6. 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.

  7. BMP4 Increases the Expression of TRPC and Basal [Ca2+]i via the p38MAPK and ERK1/2 Pathways Independent of BMPRII in PASMCs

    PubMed Central

    Tian, Lichun; Fu, Xin; Wang, Yan; Sun, Yueqian; Jiang, Qian; Lu, Wenju; Wang, Jian

    2014-01-01

    Multiple abnormalities of bone morphogenetic protein (BMPs) signaling are implicated in the process of pulmonary arterial hypertension (PAH). BMP4 plays an important role during the process of pulmonary arterial remodeling and mutant of the principle BMP4 receptor, BMP receptors II (BMPRII), is found to associate with the development of PAH. However, the likely mechanism defining the contribution of BMPRII to BMP4 mediated signaling in pulmonary arterial smooth muscle cells (PASMCs) remains comprehensively unclear. We previously found that enhanced store operated calcium entry (SOCE) and basal intracellular calcium concentration [Ca2+]i were induced by BMP4 via upregulation of TRPC1, 4 and 6 expression in PASMCs, and that BMP4 modulated TRPC channel expression through activating p38MAPK and ERK1/2 signaling pathways. In this study, BMPRII siRNA was used to knockdown BMPRII expression to investigate whether BMP4 upregulates the expression of TRPC and activating Smad1/5/8, ERK1/2 and p38MAPK pathway via BMPRII in distal PASMCs. Our results showed that knockdown of BMPRII: 1) attenuated BMP4 induced activation of P-Smad1/5/8, without altering BMP4 induced P-p38MAPK and P-ERK1/2 activation in PASMCs; 2) did not attenuate the BMP4-induced TRPC1, 4 and 6 expression; 3) did not affect BMP4-enhanced SOCE and basal [Ca2+]i. Thus, we concluded that BMP4 activated Smad1/5/8 pathway is BMPRII-dependent, while the BMP4 – ERK/p-P38 – TRPC – SOCE signaling axis are likely mediated through other receptor rather than BMPRII. PMID:25461595

  8. Tissue transglutaminase-2 promotes gastric cancer progression via the ERK1/2 pathway

    PubMed Central

    Zhou, Quan; Wu, Xiongyan; Chen, Xuehua; Li, Jianfang; Zhu, Zhenggang; Liu, Bingya; Su, Liping

    2016-01-01

    Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer. PMID:26771235

  9. Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein

    NASA Technical Reports Server (NTRS)

    Jiang, Guang-Liang; White, Charles R.; Stevens, Hazel Y.; Frangos, John A.

    2002-01-01

    Bone cells are subject to interstitial fluid flow (IFF) driven by venous pressure and mechanical loading. Rapid dynamic changes in mechanical loading cause transient gradients in IFF. The effects of pulsatile flow (temporal gradients in fluid shear) on rat UMR106 cells and rat primary osteoblastic cells were studied. Pulsatile flow induced a 95% increase in S-phase UMR106 cells compared with static controls. In contrast, ramped steady flow stimulated only a 3% increase. Similar patterns of S-phase induction were also observed in rat primary osteoblastic cells. Pulsatile flow significantly increased relative UMR106 cell number by 37 and 62% at 1.5 and 24 h, respectively. Pulsatile flow also significantly increased extracellular signal-regulated kinase (ERK1/2) phosphorylation by 418%, whereas ramped steady flow reduced ERK1/2 activation to 17% of control. Correspondingly, retinoblastoma protein was significantly phosphorylated by pulsatile fluid flow. Inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK)1/2 by U0126 (a specific MEK1/2 inhibitor) reduced shear-induced ERK1/2 phosphorylation and cell proliferation. These findings suggest that temporal gradients in fluid shear stress are potent stimuli of bone cell proliferation.

  10. Caffeic acid directly targets ERK1/2 to attenuate solar UV-induced skin carcinogenesis

    PubMed Central

    Yang, Ge; Fu, Yang; Malakhova, Margarita; Kurinov, Igor; Zhu, Feng; Yao, Ke; Li, Haitao; Chen, Hanyong; Li, Wei; Lim, Do Young; Sheng, Yuqiao; Bode, Ann M.; Dong, Ziming; Dong, Zigang

    2014-01-01

    Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in coffee and reportedly has anticancer activities. However, the underlying molecular mechanisms and targeted proteins involved in the suppression of carcinogenesis by caffeic acid are not fully understood. In this study, we report that caffeic acid significantly inhibits colony formation of human skin cancer cells and EGF-induced neoplastic transformation of HaCaT cells dose-dependently. Caffeic acid topically applied to dorsal mouse skin significantly suppressed tumor incidence and volume in a solar UV-induced skin carcinogenesis mouse model. A substantial reduction of phosphorylation in mitogen-activated protein kinase signaling was observed in mice treated with caffeic acid either before or after solar UV exposure. Caffeic acid directly interacted with ERK1/2 and inhibited ERK1/2 activities in vitro. Importantly, we resolved the co-crystal structure of ERK2 complexed with caffeic acid. Caffeic acid interacted directly with ERK2 at amino acid residues Q105, D106 and M108. Moreover, A431 cells expressing knockdown of ERK2 lost sensitivity to caffeic acid in a skin cancer xenograft mouse model. Taken together, our results suggest that caffeic acid exerts chemopreventive activity against solar UV-induced skin carcinogenesis by targeting ERK1 and 2. PMID:25104643

  11. Erk1/2 MAPK and Caldesmon Differentially Regulate Podosome Dynamics in A7r5 Vascular Smooth Muscle Cells

    PubMed Central

    Gu, Zhizhan; Kordowska, Jolanta; Williams, Geoffrey L.; Wang, C.-L. Albert; Hai, Chi-Ming

    2007-01-01

    We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained α-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells. PMID:17239373

  12. Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells

    SciTech Connect

    Gu Zhizhan; Kordowska, Jolanta; Williams, Geoffrey L.; Wang, C.-L. Albert; Hai, C.-M. . E-mail: Chi-Ming_Hai@brown.edu

    2007-03-10

    We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained {alpha}-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.

  13. GGPPS, a new EGR-1 target gene, reactivates ERK 1/2 signaling through increasing Ras prenylation.

    PubMed

    Shen, Ning; Shao, Yue; Lai, Shan-Shan; Qiao, Long; Yang, Run-Lin; Xue, Bin; Pan, Fei-Yan; Chen, Hua-Qun; Li, Chao-Jun

    2011-12-01

    Cigarette smoke activates the extracellular signal-regulated kinase (ERK) 1/2 mitogen activated-protein kinase pathway, which, in turn, is responsible for early growth response gene-1 (EGR-1) activation. Here we provide evidence that EGR-1 activation can also reactivate ERK 1/2 mitogen activated-protein kinase through a positive feedback loop through its target gene (geranylgeranyl diphosphate synthase) GGPPS. For the first time, the GGPPS gene is identified as a target of EGR-1, as EGR-1 can directly bind to the predicted consensus-binding site in the GGPPS promoter and regulate its transcription. Long-term observations show that there are two ERK 1/2 phosphorylation peaks after cigarette smoke extract stimulation in human lung epithelial Beas-2B cells. The first peak (at 10 minutes) is responsible for EGR-1 accumulation, and the second (at 4 hours) is diminished after the disruption of EGR-1 transcriptional activity. EGR-1 overexpression enhances Ras prenylation and membrane association in a GGPPS-dependent manner, and it augments ERK 1/2 activation. Likewise, a great reduction of the second peak of ERK 1/2 phosphorylation is observed during long-term cigarette smoke extract stimulation in cells where GGPPS is disrupted. Thus, we have uncovered an intricate positive feedback loop in which ERK 1/2-activated EGR-1 promotes ERK 1/2 reactivation through promoting GGPPS transcription, which might affect cigarette smoke-related lung pathological processes.

  14. Dual modulation of ERK1/2 and p38 MAP kinase activities induced by minocycline reverses the neurotoxic effects of the prion protein fragment 90-231.

    PubMed

    Corsaro, Alessandro; Thellung, Stefano; Chiovitti, Katia; Villa, Valentina; Simi, Alessandro; Raggi, Federica; Paludi, Domenico; Russo, Claudio; Aceto, Antonio; Florio, Tullio

    2009-02-01

    Several in vitro and in vivo studies addressed the identification of molecular determinants of the neuronal death induced by PrP(Sc) or related peptides. We developed an experimental model to assess PrP(Sc) neurotoxicity using a recombinant polypeptide encompassing amino acids 90-231 of human PrP (hPrP90-231) that corresponds to the protease-resistant core of PrP(Sc) identified in prion-infected brains. By means of mild thermal denaturation, we can convert hPrP90-231 from a PrP(C)-like conformation into a PrP(Sc)-like structure. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing caspase 3 and p38-dependent apoptosis, while in the native alpha-helix-rich conformation, hPrP90-231 did not induce cell toxicity. The aim of this study was to identify drugs able to block hPrP90-231 neurotoxic effects, focusing on minocycline, a tetracycline with known neuroprotective activity. hPrP90-231 caused a caspase 3-dependent apoptosis via the blockade of ERK1/2 activation and the subsequent activation of p38 MAP kinase. We propose that hPrP90-231-induced apoptosis is dependent on the inhibition of ERK1/2 responsiveness to neurotrophic factors, removing a tonic inhibition of p38 activity and resulting in caspase 3 activation. Minocycline prevented hPrP90-231-induced toxicity interfering with this mechanism: the pretreatment with this tetracycline restored ERK1/2 activity and reverted p38 and caspase 3 activities. The effects of minocycline were not mediated by the prevention of hPrP90-231 structural changes or cell internalization (differently from Congo Red). In conclusion, minocycline elicits anti-apoptotic effects against the neurotoxic activity of hPrP90-231 and these effects are mediated by opposite modulation of ERK1/2 and p38 MAP kinase activities.

  15. Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

    SciTech Connect

    Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique; Mikalsen, Svein-Ole; Slipicevic, Ana; Florenes, Vivi Ann . E-mail: v.a.florenes@labmed.uio.no

    2005-04-01

    The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.

  16. A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Pooran, Milad; Li, Chai-Fei; Luzzio, Chris; Hughes-Fulford, Millie

    2003-01-01

    Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway.

  17. A novel matrix metalloproteinase-2 inhibitor triazolylmethyl aziridine reduces melanoma cell invasion, angiogenesis and targets ERK1/2 phosphorylation.

    PubMed

    Romanchikova, Nadezhda; Trapencieris, Pēteris; Zemītis, Jānis; Turks, Māris

    2014-12-01

    A novel matrix metalloproteinase-2 (MMP-2) inhibitor JaZ-30, which belongs to the class of C(2)-monosubstituted aziridine - aryl-1,2,3-triazole conjugates, was developed. MTT and crystal violet assays were used to determine cytotoxicity- IC(50) values of compound JaZ-30 on melanoma cell line B16 4A5. Our study proves the anti-cancer properties of JaZ-30 with a wide spectrum of activities. JaZ-30 was revealed as selective inhibitor of matrix metalloproteinase-2. JaZ-30-mediated decrease of Vascular Endothelial Growth Factor (VEGF) secretion results in inhibition of angiogenesis, performed with the human umbilical vein endothelial cell line (HUVEC-2) on Matrigel. A novel inhibitor decreases invasive properties of melanoma cells measured in Matrigel chambers assay. JaZ-30 downregulates phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in melanoma cells stimulated by phorbol-12-myristate-13-acetate (PMA). Our findings propose a novel MMP-2 inhibitor JaZ-30 as an attractive potential agent for melanoma treatment.

  18. Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

    SciTech Connect

    Yonezawa, Tomo Haga, Satoshi; Kobayashi, Yosuke; Katoh, Kazuo; Obara, Yoshiaki

    2008-03-21

    GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca{sup 2+} concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.

  19. The antitumor effect of TIG3 in liver cancer cells is involved in ERK1/2 inhibition.

    PubMed

    Xu, Yan; Chen, Ting; Liao, Degui; Wu, Xiaoqin; Zhong, Yun; Liu, Shiming; Yang, Hui; Nie, Yuqiang

    2016-08-01

    Tazarotene-induced gene 3 (TIG3) was first characterized in tazarotene-treated human keratinocytes and identified as a retinoic acid responder gene, an important mediator of antitumor effects by retinoids. In this study, we aim to investigate the inhibitory effect of TIG3 on the growth of liver cancer and explore its underlying mechanism. Human hepatocellular carcinoma (HCC) Hep3B cells were transfected with plasmid GV141 carrying full-length TIG3 complementary DNA (cDNA). The effects of TIG3 on cell proliferation, apoptosis, and migration were determined in vitro. The suppressor effect of TIG3 on tumor growth was evaluated in vivo in a nude mouse HCC model. We observed that TIG3 expression is decreased in the Hep3B cell line as well as primary HCC tumors, and TIG3 expression inversely correlates with Ki-67 expression. Overexpression of TIG3 suppresses tumor growth in HCC both in vitro and in vivo via ERK1/2 inhibition by promoting apoptosis and inhibiting proliferation and migration. These findings identify TIG3 as an attractive therapeutic target for HCC.

  20. Inhibition of ERK1/2 down-regulates the Hippo/YAP signaling pathway in human NSCLC cells

    PubMed Central

    Xu, Zhidong; Dai, Yuyuan; Liu, Shu; Mao, Jian-Hua; Tetsu, Osamu; Li, Hui; Jablons, David M.; You, Liang

    2015-01-01

    Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC). Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3′UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and transcriptional activity of the Hippo pathway in NSCLC cell lines. Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells. PMID:25738359

  1. Tiotropium bromide suppresses smoke inhalation and burn injury-induced ERK 1/2 and SMAD 2/3 signaling in sheep bronchial submucosal glands.

    PubMed

    Jacob, Sam; Zhu, Yong; Asmussen, Sven; Ito, Hiroshi; Herndon, David N; Enkhbaatar, Perenlei; Hawkins, Hal K; Cox, Robert A

    2014-05-01

    The effects of tiotropium bromide on ERK 1/2, SMAD 2/3 and NFκB signaling in bronchial submucosal gland (SMG) cells of sheep after smoke inhalation and burn injury (S + B) were studied. We hypothesized that tiotropium would modify intracellular signaling processes within SMG cells after injury. Bronchial tissues were obtained from uninjured (sham, n = 6), S + B injured sheep 48 h after injury (n = 6), and injured sheep nebulized with tiotropium (n = 6). The percentage (mean ± SD) of cells showing nuclear localization of phosphorylated ERK 1/2, pSMAD 2/3, and NFκB (p65) was determined by immunohistochemistry. Nuclear pERK 1/2 staining was increased in injured animals as compared to sham, (66 ± 20 versus 14 ± 9), p = 0.0022, as was nuclear pSMAD, 84 ± 10 versus 20 ± 10, p = 0.0022. There was a significant decrease in pERK 1/2 labeling in the tiotropium group compared to the injured group (31 ± 20 versus 66 ± 20, p = 0.013), and also a decrease in pSMAD labeling, 62 ± 17 versus 84 ± 10, p = 0.04. A significant increase for NFκB (p65) was noted in injured animals as compared to sham (73 ± 16 versus 7 ± 6, p = 0.0022). Tiotropium-treated animals showed decreased p65 labeling as compared to injured (35 ± 17 versus 74 ± 16, p = 0.02). The decrease in nuclear expression of pERK, pSMAD and NFκB molecules in SMG cells with tiotropium treatment is suggestive that their activation after injury is mediated in part through muscarinic receptors.

  2. PKC-α contributes to high NaCl-induced activation of NFAT5 (TonEBP/OREBP) through MAPK ERK1/2.

    PubMed

    Wang, Hong; Ferraris, Joan D; Klein, Janet D; Sands, Jeff M; Burg, Maurice B; Zhou, Xiaoming

    2015-01-15

    High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser(591) on Src homology 2 domain-containing phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591. PMID:25391900

  3. Axl mediates acquired resistance of head and neck cancer cells to the epidermal growth factor receptor inhibitor erlotinib.

    PubMed

    Giles, Keith M; Kalinowski, Felicity C; Candy, Patrick A; Epis, Michael R; Zhang, Priscilla M; Redfern, Andrew D; Stuart, Lisa M; Goodall, Gregory J; Leedman, Peter J

    2013-11-01

    Elevated expression and activity of the epidermal growth factor receptor (EGFR) is associated with development and progression of head and neck cancer (HNC) and a poor prognosis. Clinical trials with EGFR tyrosine kinase inhibitors (e.g., erlotinib) have been disappointing in HNC. To investigate the mechanisms mediating resistance to these agents, we developed an HNC cell line (HN5-ER) with acquired erlotinib resistance. In contrast to parental HN5 HNC cells, HN5-ER cells exhibited an epithelial-mesenchymal (EMT) phenotype with increased migratory potential, reduced E-cadherin and epithelial-associated microRNAs (miRNA), and elevated vimentin expression. Phosphorylated receptor tyrosine kinase profiling identified Axl activation in HN5-ER cells. Growth and migration of HN5-ER cells were blocked with a specific Axl inhibitor, R428, and R428 resensitized HN5-ER cells to erlotinib. Microarray analysis of HN5-ER cells confirmed the EMT phenotype associated with acquired erlotinib resistance, and identified activation of gene expression associated with cell migration and inflammation pathways. Moreover, increased expression and secretion of interleukin (IL)-6 and IL-8 in HN5-ER cells suggested a role for inflammatory cytokine signaling in EMT and erlotinib resistance. Expression of the tumor suppressor miR-34a was reduced in HN5-ER cells and increasing its expression abrogated Axl expression and reversed erlotinib resistance. Finally, analysis of 302 HNC patients revealed that high tumor Axl mRNA expression was associated with poorer survival (HR = 1.66, P = 0.007). In summary, our results identify Axl as a key mediator of acquired erlotinib resistance in HNC and suggest that therapeutic inhibition of Axl by small molecule drugs or specific miRNAs might overcome anti-EGFR therapy resistance. PMID:24026012

  4. The class II transactivator (CIITA) is regulated by post-translational modification cross-talk between ERK1/2 phosphorylation, mono-ubiquitination and Lys63 ubiquitination.

    PubMed

    Morgan, Julie E; Shanderson, Ronald L; Boyd, Nathaniel H; Cacan, Ercan; Greer, Susanna F

    2015-06-19

    The class II transactivator (CIITA) is known as the master regulator for the major histocompatibility class II (MHC II) molecules. CIITA is dynamically regulated through a series of intricate post-translational modifications (PTMs). CIITA's role is to initiate transcription of MHC II genes, which are responsible for presenting extracellular antigen to CD4(+) T-cells. In the present study, we identified extracellular signal-regulated kinase (ERK)1/2 as the kinase responsible for phosphorylating the regulatory site, Ser(280), which leads to increased levels of mono-ubiquitination and an overall increase in MHC II activity. Further, we identify that CIITA is also modified by Lys(63)-linked ubiquitination. Lys(63) ubiquitinated CIITA is concentrated in the cytoplasm and following activation of ERK1/2, CIITA phosphorylation occurs and Lys=ubiquitinated CIITA translocates to the nucleus. CIITA ubiquitination and phosphorylation perfectly demonstrates how CIITA location and activity is regulated through PTM cross-talk. Identifying CIITA PTMs and understanding how they mediate CIITA regulation is necessary due to the critical role CIITA has in the initiation of the adaptive immune response.

  5. TcpC protein from E. coli Nissle improves epithelial barrier function involving PKCζ and ERK1/2 signaling in HT-29/B6 cells.

    PubMed

    Hering, N A; Richter, J F; Fromm, A; Wieser, A; Hartmann, S; Günzel, D; Bücker, R; Fromm, M; Schulzke, J D; Troeger, H

    2014-03-01

    The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing effects in the intestine. In this study, the mechanisms of barrier modulation by EcN were studied in the human epithelial HT-29/B6 cell culture model.EcN supernatant increased transepithelial resistance (TER) and reduced permeability to mannitol because of sealing of the paracellular passage pathway as revealed by two-path impedance spectroscopy. This increase in TER was attributed to the TcpC protein of EcN. TcpC induced protein kinase C-ζ (PKCζ) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which in turn resulted in upregulation of the barrier-forming tight junction protein claudin-14. By specific silencing of protein expression by small interfering RNA (siRNA), the sealing function of claudin-14 was confirmed. In conclusion, the TcpC protein of EcN affects innate immunity by improving intestinal barrier function through upregulation of claudin-14 via PKCζ and ERK1/2 signaling. PMID:23900194

  6. Dopamine D1/D5 receptor signaling regulates synaptic cooperation and competition in hippocampal CA1 pyramidal neurons via sustained ERK1/2 activation

    PubMed Central

    Shivarama Shetty, Mahesh; Gopinadhan, Suma

    2016-01-01

    ABSTRACT Synaptic cooperation and competition are important components of synaptic plasticity that tune synapses for the formation of associative long‐term plasticity, a cellular correlate of associative long‐term memory. We have recently reported that coincidental activation of weak synapses within the vicinity of potentiated synapses will alter the cooperative state of synapses to a competitive state thus leading to the slow decay of long‐term plasticity, but the molecular mechanism underlying this is still unknown. Here, using acute hippocampal slices of rats, we have examined how increasing extracellular dopamine concentrations interact and/or affect electrically induced long‐term potentiation (LTP) in the neighboring synapses. We demonstrate that D1/D5‐receptor‐mediated potentiation at the CA1 Schaffer collateral synapses differentially regulates synaptic co‐operation and competition. Further investigating the molecular players involved, we reveal an important role for extracellular signal‐regulated kinases‐1 and 2 (ERK1/2) as signal integrators and dose‐sensors. Interestingly, a sustained activation of ERK1/2 pathway seems to be involved in the differential regulation of synaptic associativity. The concentration‐dependent effects of the modulatory transmitter, as demonstrated for dopaminergic signaling in the present study, might offer additional computational power by fine tuning synaptic associativity processes for establishing long‐term associative memory in neural networks. © 2015 The Authors Hippocampus Published by Wiley Periodicals, Inc. PMID:26194339

  7. Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ape1/Ref-1) is essential for IL-21-induced signal transduction through ERK1/2 pathway.

    PubMed

    Juliana, Farha M; Nara, Hidetoshi; Onoda, Tadashi; Rahman, Mizanur; Araki, Akemi; Jin, Lianjin; Fujii, Hodaka; Tanaka, Nobuyuki; Hoshino, Tomoaki; Asao, Hironobu

    2012-04-13

    IL-21 is a pleiotropic cytokine that regulates T-cell and B-cell differentiation, NK-cell activation, and dendritic cell functions. IL-21 activates the JAK-STAT, ERK, and PI3K pathways. We report here that Ape1/Ref-1 has an essential role in IL-21-induced cell growth signal transduction. Overexpression of Ape1/Ref-1 enhances IL-21-induced cell proliferation, but it is suppressed by overexpressing an N-terminal deletion mutant of Ape1/Ref-1 that lacks the redox domain. Furthermore, knockdown of the Ape1/Ref-1 mRNA dramatically compromises IL-21-induced ERK1/2 activation and cell proliferation with increasing cell death. These impaired activities are recovered by the re-expression of Ape1/Ref-1 in the knockdown cells. Our findings are the first demonstration that Ape1/Ref-1 is an indispensable molecule for the IL-21-mediated signal transduction through ERK1/2 activation.

  8. Methylcobalamin promotes proliferation and migration and inhibits apoptosis of C2C12 cells via the Erk1/2 signaling pathway

    SciTech Connect

    Okamoto, Michio; Tanaka, Hiroyuki; Okada, Kiyoshi; Kuroda, Yusuke; Nishimoto, Shunsuke; Murase, Tsuyoshi; Yoshikawa, Hideki

    2014-01-17

    Highlights: •Methylcobalamin activated the Erk1/2 signaling pathway in C2C12 cells. •Methylcobalamin promoted the proliferation and migration in C2C12 cells. •C2C12 cell apoptosis during differentiation was inhibited by methylcobalamin. -- Abstract: Methylcobalamin (MeCbl) is a vitamin B12 analog that has some positive effects on peripheral nervous disorders. Although some previous studies revealed the effects of MeCbl on neurons, its effect on the muscle, which is the final target of motoneuron axons, remains to be elucidated. This study aimed to determine the effect of MeCbl on the muscle. We found that MeCbl promoted the proliferation and migration of C2C12 myoblasts in vitro and that these effects are mediated by the Erk1/2 signaling pathway without affecting the activity of the Akt signaling pathway. We also demonstrated that MeCbl inhibits C2C12 cell apoptosis during differentiation. Our results suggest that MeCbl has beneficial effects on the muscle in vitro. MeCbl administration may provide a novel therapeutic approach for muscle injury or degenerating muscle after denervation.

  9. Isoniazid prevents Nrf2 translocation by inhibiting ERK1 phosphorylation and induces oxidative stress and apoptosis

    PubMed Central

    Verma, Ajeet Kumar; Yadav, Arti; Dewangan, Jayant; Singh, Sarvendra Vikram; Mishra, Manisha; Singh, Pradhyumna Kumar; Rath, Srikanta Kumar

    2015-01-01

    Isoniazid is used either alone or in combination with other drugs for the treatment of tuberculosis. It is also used for the prevention of tuberculosis. Chronic treatment of Isoniazid may cause severe liver damage leading to acute liver failure. The mechanism through which Isoniazid causes liver damage is investigated. Isoniazid treatment generates reactive oxygen species and induces apoptosis in Hep3B cells. It induces antioxidative and apoptotic genes leading to increase in mRNA expression and protein levels in Hep3B cells. Whole genome expression analysis of Hep3B cells treated with Isoniazid has resulted in differential expression of various genes playing prime role in regulation of apoptotic, antioxidative, DNA damage, cell signaling, cell proliferation and differentiation pathways. Isoniazid increased cytosolic Nrf2 protein level while decreased nuclear Nrf2 protein level. It also decreased ERK1 phosphorylation and treatment of Hep3B cells with ERK inhibitor followed by Isoniazid resulting in increased apoptosis in these cells. Two dimensional gel electrophoresis results have also shown differential expression of various protein species including heat shock proteins, proteins playing important role in oxidative stress, DNA damage, apoptosis, cell proliferation and differentiation. Results suggest that Isoniazid induces apoptosis through oxidative stress and also prevents Nrf2 translocation into the nucleus by reducing ERK1 phosphorylation thus preventing cytoprotective effect. PMID:26202867

  10. Overexpression of Raf-1 and ERK1/2 in sacral chordoma and association with tumor recurrence

    PubMed Central

    Zhang, Kai; Chen, Hao; Zhang, Bin; Sun, Jiajia; Lu, Jian; Chen, Kangwu; Yang, Huilin

    2015-01-01

    Chordoma is a rare and low-malignant neoplasm which is considered to arise from notochord remnants. Due to its large resistance to chemotherapy and radiotherapy, surgical resection so far is the prior treatment for chordoma. However, the recurrence rate is high even after complete surgical resection. Recently, targeted cancer therapy has been demonstrated to be effective in several other tumors, while the related research on chordoma is rare. Mitogen-activated protein kinase signaling pathway is acknowledged to participate in tumor development, in which Raf-1 and extracellular regulated protein kinase 1/2 (ERK1/2) play vital roles. In this study, we evaluated the expression of Raf-1 and ERK1/2 by immunohistochemical staining in 42 chordoma tissue and 16 distant normal tissue. Moreover, we also investigated the correlations of Raf-1 and ERK1/2 expression with clinical features in sacral chordoma. Expression of Raf-1 and ERK1/2 was both significantly higher in sacral chordoma tissue than distant normal tissue (P = 0.008, P = 0.019). Raf-1 positive expression was related to surrounding muscle invasion (P = 0.032) and chordoma recurrence (P = 0.002), but the results did not indicate any association with patients’ age, gender, tumor size and location. ERK1/2 was associated with tumor size (P = 0.044) instead of other clinical factors (P > 0.05). Spearman correlation test showed close relation between ERK1/2 and Raf-1 (P = 0.001, r = 0.518). Kaplan–Meier survival Curve and log-rank test showed that Raf-1 positive expression was associated with shorter continuous disease-free survival time (CDFS) (P = 0.001), while ERK1/2 had no relation to CDFS (P = 0.961). Conclusively, Raf-1 may be an important biomarker in predicting the prognosis of chordoma patients. PMID:25755752

  11. [The expression of MKP-1 and p-ERK(1/2) in primary ovarian epithelial tumor tissues].

    PubMed

    Zhou, Jian Wei; Gan, Ning Yue; Zhang, Wei Jiang

    2009-06-01

    To investigate the expression of mitogen activated protein kinase phosphatase-1 (MKP-1) and phosphorylation extracellular signal-regulated kinases (p-ERK(1/2)) in primary ovarian epithelial tumor tissues, and provide experiment's foundation on the new treatment in ovarian cancer. Expression of MKP-1 and p-ERK(1/2) in tissues from 64 patients with primary ovarian epithelial tumor, 35 patients with ovarian epithelial bordline tumor, 32 patients with ovarian epithelial benign tumor and 26 normal ovarian tissues was detected by immunohistochemistry. Western-blot was also used for detecting the expression of MKP-1 and p-ERK(1/2) protein in these tissues. Immunohistochemistry and Western-blot assay showed that the expression of MKP-1 was gradually decreased in normal ovarian tissues, benign tumor, bordline tumor and carcinoma respectively, and there were significant differences among them (P < 0.01). The MKP-1 expression level in the carcinoma tissues of stage III/IV patients was significantly lower than that of stage I/II patients. However, the expression of p-ERK(1/2) was gradually increased in normal ovarian tissues, benign tumor, bordline tumor and carcinoma respectively, and there were also significant differences among them (P < 0.01), the p-ERK(1/2) expression level in the carcinoma tissues of stage III/IV patients was significantly higher than that of stage I/II patients. Expression of MKP-1 and p-ERK(1/2) in same ovarian carcinoma tissues detected by immunohistochemistry and Western-blot assay showed significant negative correlation (r = -0.90, P < 0.01 and r = -0.78, P < 0.01 respectively). The expression changes of MKP-1 and ERKs may play a role in the development of ovarian carcinoma. The abnormal expression of MKP-1 and p-ERK(1/2) probably assists in promoting the development and progression of ovarian carcinoma.

  12. The MAPK ERK5, but not ERK1/2, inhibits the progression of monocytic phenotype to the functioning macrophage

    SciTech Connect

    Wang, Xuening; Pesakhov, Stella; Harrison, Jonathan S; Kafka, Michael; Danilenko, Michael; Studzinski, George P

    2015-01-01

    Intracellular signaling pathways present targets for pharmacological agents with potential for treatment of neoplastic diseases, with some disease remissions already recorded. However, cellular compensatory mechanisms usually negate the initial success. For instance, attempts to interrupt aberrant signaling downstream of the frequently mutated ras by inhibiting ERK1/2 has shown only limited usefulness for cancer therapy. Here, we examined how ERK5, that overlaps the functions of ERK1/2 in cell proliferation and survival, functions in a manner distinct from ERK1/2 in human AML cells induced to differentiate by 1,25D-dihydroxyvitamin D{sub 3} (1,25D). Using inhibitors of ERK1/2 and of MEK5/ERK5 at concentrations specific for each kinase in HL60 and U937 cells, we observed that selective inhibition of the kinase activity of ERK5, but not of ERK1/2, in the presence of 1,25D resulted in macrophage-like cell morphology and enhancement of phagocytic activity. Importantly, this was associated with increased expression of the macrophage colony stimulating factor receptor (M-CSFR), but was not seen when M-CSFR expression was knocked down. Interestingly, inhibition of ERK1/2 led to activation of ERK5 in these cells. Our results support the hypothesis that ERK5 negatively regulates the expression of M-CSFR, and thus has a restraining function on macrophage differentiation. The addition of pharmacological inhibitors of ERK5 may influence trials of differentiation therapy of AML. - Highlights: • ERK5 has at least some functions in AML cells which are distinct from those of ERK1/2. • ERK5 activity negatively controls the expression of M-CSFR. • ERK5 retards the progression of differentiation from monocyte to functional macrophage.

  13. Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)*

    PubMed Central

    Chiluiza, David; Krishna, Sneha; Schumacher, Valérie A.; Schlöndorff, Johannes

    2013-01-01

    Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) gene are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS). The mechanisms whereby abnormal TRPC6 activity results in proteinuria remain unknown. The ERK1/2 MAPKs are activated in glomeruli and podocytes in several proteinuric disease models. We therefore examined whether FSGS-associated mutations in TRPC6 result in activation of these kinases. In 293T cells and cultured podocytes, overexpression of gain-of-function TRPC6 mutants resulted in increased ERK1/2 phosphorylation, an effect dependent upon channel function. Pharmacologic inhibitor studies implicated several signaling mediators, including calmodulin and calcineurin, supporting the importance of TRPC6-mediated calcium influx in this process. Through medium transfer experiments, we uncovered two distinct mechanisms for ERK activation by mutant TRPC6, a cell-autonomous, EGF receptor-independent mechanism and a non-cell-autonomous mechanism involving metalloprotease-mediated release of a presumed EGF receptor ligand. The inhibitors KN-92 and H89 were able to block both pathways in mutant TRPC6 expressing cells as well as the prolonged elevation of intracellular calcium levels upon carbachol stimulation seen in these cells. However, these effects appear to be independent of their effects on calcium/calmodulin-dependent protein kinase II and PKA, respectively. Phosphorylation of Thr-70, Ser-282, and Tyr-31/285 were not necessary for ERK activation by mutant TRPC6, although a phosphomimetic TRPC6 S282E mutant was capable of ERK activation. Taken together, these results identify two pathways downstream of mutant TRPC6 leading to ERK activation that may play a role in the development of FSGS. PMID:23645677

  14. ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation

    PubMed Central

    de Jong, Petrus R.; Taniguchi, Koji; Harris, Alexandra R.; Bertin, Samuel; Takahashi, Naoki; Duong, Jen; Campos, Alejandro D.; Powis, Garth; Corr, Maripat; Karin, Michael; Raz, Eyal

    2016-01-01

    The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC. PMID:27187615

  15. Mathematical modeling reveals the functional implications of the different nuclear shuttling rates of Erk1 and Erk2

    NASA Astrophysics Data System (ADS)

    Harrington, Heather A.; Komorowski, Michał; Beguerisse-Díaz, Mariano; Ratto, Gian Michele; Stumpf, Michael P. H.

    2012-06-01

    The mitogen-activated protein kinase (MAPK) family of proteins is involved in regulating cellular fates such as proliferation, differentiation and apoptosis. In particular, the dynamics of the Erk/Mek system, which has become the canonical example for MAPK signaling systems, have attracted considerable attention. Erk is encoded by two genes, Erk1 and Erk2, that until recently had been considered equivalent as they differ only subtly at the sequence level. However, these proteins exhibit radically different trafficking between cytoplasm and nucleus and this fact may have functional implications. Here we use spatially resolved data on Erk1/2 to develop and analyze spatio-temporal models of these cascades, and we discuss how sensitivity analysis can be used to discriminate between mechanisms. Our models elucidate some of the factors governing the interplay between signaling processes and the Erk1/2 localization in different cellular compartments, including competition between Erk1 and Erk2. Our approach is applicable to a wide range of signaling systems, such as activation cascades, where translocation of molecules occurs. Our study provides a first model of Erk1 and Erk2 activation and their nuclear shuttling dynamics, revealing a role in the regulation of the efficiency of nuclear signaling.

  16. Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs.

    PubMed

    Tee, Wee-Wei; Shen, Steven S; Oksuz, Ozgur; Narendra, Varun; Reinberg, Danny

    2014-02-13

    Erk1/2 activation contributes to mouse ES cell pluripotency. We found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation.

  17. ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation.

    PubMed

    de Jong, Petrus R; Taniguchi, Koji; Harris, Alexandra R; Bertin, Samuel; Takahashi, Naoki; Duong, Jen; Campos, Alejandro D; Powis, Garth; Corr, Maripat; Karin, Michael; Raz, Eyal

    2016-05-17

    The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.

  18. Aristolochia Manshuriensis Kom Inhibits Adipocyte Differentiation by Regulation of ERK1/2 and Akt Pathway

    PubMed Central

    Kwak, Dong Hoon; Lee, Ji-Hye; Kim, Taesoo; Ahn, Hyo Sun; Cho, Won-Kyung; Ha, Hyunil; Hwang, Youn-Hwan; Ma, Jin Yeul

    2012-01-01

    Aristolochia manshuriensis Kom (AMK) is a traditional medicinal herb used for the treatment of arthritis, rheumatism, hepatitis, and anti-obesity. Because of nephrotoxicity and carcinogenicity of AMK, there are no pharmacological reports on anti-obesity potential of AMK. Here, we showed AMK has an inhibitory effect on adipocyte differentiation of 3T3-L1 cells along with significantly decrease in the lipid accumulation by downregulating several adipocyte-specific transcription factors including peroxisome proliferation-activity receptor γ (PPAR-γ), CCAAT/enhancer binding protein α (C/EBP-α) and C/EBP-β, which are critical for adipogenesis in vitro. AMK also markedly activated the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway including Ras, Raf1, and mitogen-activated protein kinase kinase 1 (MEK1), and significantly suppressed Akt pathway by inhibition of phosphoinositide-dependent kinase 1 (PDK1). Aristolochic acid (AA) and ethyl acetate (EtOAc) fraction of AMK with AA were significantly inhibited TG accumulation, and regulated two pathway (ERK1/2 and Akt) during adipocyte differentiation, and was not due to its cytotoxicity. These two pathways were upstream of PPAR-γ and C/EBPα in the adipogenesis. In addition, gene expressions of secreting factors such as fatty acid synthase (FAS), adiponectin, lipopreotein lipase (LPL), and aP2 were significantly inhibited by treatment of AMK during adipogenesis. We used the high-fat diet (HFD)-induced obesity mouse model to determine the inhibitory effects of AMK on obesity. Oral administration of AMK (62.5 mg/kg/day) significantly decreased the fat tissue weight, total cholesterol (TC), and low density lipoprotein-cholesterol (LDL-C) concentration in the blood. The results of this study suggested that AMK inhibited lipid accumulation by the down-regulation of the major transcription factors of the adipogensis pathway including PPAR-γ and C/EBP-α through regulation of Akt pathway and ERK 1

  19. Prostaglandin E2 transactivates the colony-stimulating factor-1 receptor and synergizes with colony-stimulating factor-1 in the induction of macrophage migration via the mitogen-activated protein kinase ERK1/2.

    PubMed

    Digiacomo, Graziana; Ziche, Marina; Dello Sbarba, Persio; Donnini, Sandra; Rovida, Elisabetta

    2015-06-01

    Prostaglandin E2 (PGE2), a key mediator of immunity, inflammation, and cancer, acts through 4 G-protein-coupled E-prostanoid receptors (EPs 1-4). Crosstalk between EPs and receptor tyrosine kinases also occurs. Colony-stimulating factor-1 receptor (CSF-1R) is an RTK that sustains the survival, proliferation, and motility of monocytes/macrophages, which are an essential component of innate immunity and cancer development. The aim of this study was to investigate on a possible crosstalk between EP and CSF-1R. In BAC1.2F5 and RAW264.7 murine macrophages, CSF-1 (EC₅₀ = 18.1 and 10.2 ng/ml, respectively) and PGE2 (EC₅₀ = 1.5 and 5.5 nM, respectively) promoted migration. PGE2 induced rapid CSF-1R phosphorylation that was dependent on Src family kinases (SFKs). CSF-1R inhibition reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that CSF-1R plays a role in PGE2-mediated immunoregulation. EP4 appeared responsible for functional PGE2/CSF-1R crosstalk. Furthermore, PGE2 synergized with CSF-1 in inducing ERK1/2 phosphorylation and macrophage migration. ERK1/2 inhibition completely blocked migration induced by the combination CSF-1/PGE2. CSF-1/PGE2 functional interaction with respect to migration also occurred in bone marrow-derived murine macrophages (EC₅₀ CSF-1, 6.7 ng/ml; EC₅₀ PGE2, 16.7 nM). These results indicated that PGE2 transactivates CSF-1R and synergizes with its signaling at ERK1/2 level in promoting macrophage migration.

  20. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

    PubMed Central

    Noll, Elisa M.; Eisen, Christian; Stenzinger, Albrecht; Espinet, Elisa; Muckenhuber, Alexander; Klein, Corinna; Vogel, Vanessa; Klaus, Bernd; Nadler, Wiebke; Rösli, Christoph; Lutz, Christian; Kulke, Michael; Engelhardt, Jan; Zickgraf, Franziska M.; Espinosa, Octavio; Schlesner, Matthias; Jiang, Xiaoqi; Kopp-Schneider, Annette; Neuhaus, Peter; Bahra, Marcus; Sinn, Bruno V.; Eils, Roland; Giese, Nathalia A.; Hackert, Thilo; Strobel, Oliver; Werner, Jens; Büchler, Markus W.; Weichert, Wilko; Trumpp, Andreas; Sprick, Martin R.

    2016-01-01

    Although subtypes of pancreatic ductal adenocarcinoma (PDAC) were described, this malignancy is clinically still treated as a single disease. Here, we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Individuals bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4 alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. PMID:26855150

  1. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.

    PubMed

    Noll, Elisa M; Eisen, Christian; Stenzinger, Albrecht; Espinet, Elisa; Muckenhuber, Alexander; Klein, Corinna; Vogel, Vanessa; Klaus, Bernd; Nadler, Wiebke; Rösli, Christoph; Lutz, Christian; Kulke, Michael; Engelhardt, Jan; Zickgraf, Franziska M; Espinosa, Octavio; Schlesner, Matthias; Jiang, Xiaoqi; Kopp-Schneider, Annette; Neuhaus, Peter; Bahra, Marcus; Sinn, Bruno V; Eils, Roland; Giese, Nathalia A; Hackert, Thilo; Strobel, Oliver; Werner, Jens; Büchler, Markus W; Weichert, Wilko; Trumpp, Andreas; Sprick, Martin R

    2016-03-01

    Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. PMID:26855150

  2. cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1.

    PubMed Central

    Csar, X F; Ward, A C; Hoffmann, B W; Guy, G G; Hamilton, J A

    1997-01-01

    The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization. PMID:9078246

  3. cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1.

    PubMed

    Csar, X F; Ward, A C; Hoffmann, B W; Guy, G G; Hamilton, J A

    1997-02-15

    The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.

  4. IL-17A activates ERK1/2 and enhances differentiation of oligodendrocyte progenitor cells.

    PubMed

    Rodgers, Jane M; Robinson, Andrew P; Rosler, Elen S; Lariosa-Willingham, Karen; Persons, Rachael E; Dugas, Jason C; Miller, Stephen D

    2015-05-01

    Inflammatory signals present in demyelinated multiple sclerosis lesions affect the reparative remyelination process conducted by oligodendrocyte progenitor cells (OPCs). Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 have differing effects on the viability and growth of OPCs, however the effects of IL-17A are largely unknown. Primary murine OPCs were stimulated with IL-17A and their viability, proliferation, and maturation were assessed in culture. IL-17A-stimulated OPCs exited the cell cycle and differentiated with no loss in viability. Expression of the myelin-specific protein, proteolipid protein, increased in a cerebellar slice culture assay in the presence of IL-17A. Downstream, IL-17A activated ERK1/2 within 15 min and induced chemokine expression in 2 days. These results demonstrate that IL-17A exposure stimulates OPCs to mature and participate in the inflammatory response.

  5. A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt.

    PubMed

    Olafsdottir, Astridur; Thorlacius, Gudny Ella; Omarsdottir, Sesselja; Olafsdottir, Elin Soffia; Vikingsson, Arnor; Freysdottir, Jona; Hardardottir, Ingibjorg

    2014-09-25

    Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent. PMID:24877713

  6. Elevated P75NTR expression causes death of engrailed-deficient midbrain dopaminergic neurons by Erk1/2 suppression

    PubMed Central

    Alavian, Kambiz N; Sgadò, Paola; Alberi, Lavinia; Subramaniam, Srinivasa; Simon, Horst H

    2009-01-01

    Background The homeodomain transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic (mesDA) neurons in a cell-autonomous and gene-dose-dependent manner. Homozygote mutant mice, deficient of both genes (En1-/-;En2-/-), die at birth and exhibit a loss of all mesDA neurons by mid-gestation. In heterozygote animals (En1+/-;En2-/-), which are viable and fertile, postnatal maintenance of the nigrostriatal dopaminergic system is afflicted, leading to a progressive degeneration specific to this subpopulation and Parkinson's disease-like molecular and behavioral deficits. Results In this work, we show that the dose of Engrailed is inversely correlated to the expression level of the pan-neurotrophin receptor gene P75NTR (Ngfr). Loss of mesDA neurons in the Engrailed-null mutant embryos is caused by elevated expression of this neurotrophin receptor: Unusually, in this case, the cell death signal of P75NTR is mediated by suppression of Erk1/2 (extracellular-signal-regulated kinase 1/2) activity. The reduction in expression of Engrailed, possibly related to the higher levels of P75NTR, also decreases mitochondrial stability. In particular, the dose of Engrailed determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to inhibition of the anti-apoptotic members of the Bcl-2 family of proteins. Conclusion Our study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult. The similarities to the disease etiology in combination with the nigral phenotype of En1+/-;En2-/- mice suggests that haplotype variations in the Engrailed genes and/or P75NTR that alter their expression levels could, in part, determine susceptibility to Parkinson's disease. PMID:19291307

  7. Angiotensin II Signaling in Human Preadipose Cells: Participation of ERK1,2-Dependent Modulation of Akt

    PubMed Central

    Dünner, Natalia; Quezada, Carolina; Berndt, F. Andrés; Cánovas, José; Rojas, Cecilia V.

    2013-01-01

    The renin-angiotensin system expressed in adipose tissue has been implicated in the modulation of adipocyte formation, glucose metabolism, triglyceride accumulation, lipolysis, and the onset of the adverse metabolic consequences of obesity. As we investigated angiotensin II signal transduction mechanisms in human preadipose cells, an interplay of extracellular-signal-regulated kinases 1 and 2 (ERK1,2) and Akt/PKB became evident. Angiotensin II caused attenuation of phosphorylated Akt (p-Akt), at serine 473; the p-Akt/Akt ratio decreased to 0.5±0.2-fold the control value without angiotensin II (p<0.001). Here we report that the reduction of phosphorylated Akt associates with ERK1,2 activities. In the absence of angiotensin II, inhibition of ERK1,2 activation with U0126 or PD98059 resulted in a 2.1±0.5 (p<0.001) and 1.4±0.2-fold (p<0.05) increase in the p-Akt/Akt ratio, respectively. In addition, partial knockdown of ERK1 protein expression by the short hairpin RNA technique also raised phosphorylated Akt in these cells (the p-Akt/Akt ratio was 1.5±0.1-fold the corresponding control; p<0.05). Furthermore, inhibition of ERK1,2 activation with U0126 prevented the reduction of p-Akt/Akt by angiotensin II. An analogous effect was found on the phosphorylation status of Akt downstream effectors, the forkhead box (Fox) proteins O1 and O4. Altogether, these results indicate that angiotensin II signaling in human preadipose cells involves an ERK1,2-dependent attenuation of Akt activity, whose impact on the biological functions under its regulation is not fully understood. PMID:24098385

  8. THE MAPK ERK5, BUT NOT ERK1/2, INHIBITS THE PROGRESSION OF MONOCYTIC PHENOTYPE TO THE FUNCTIONING MACROPHAGE

    PubMed Central

    Wang, Xuening; Pesakhov, Stella; Harrison, Jonathan S; Kafka, Michael; Danilenko, Michael; Studzinski, George P

    2014-01-01

    Intracellular signaling pathways present targets for pharmacological agents with potential for treatment of neoplastic diseases, with some disease remissions already recorded. However, cellular compensatory mechanisms usually negate the initial success. For instance, attempts to interrupt aberrant signaling downstream of the frequently mutated ras by inhibiting ERK1/2 has shown only limited usefulness for cancer therapy. Here, we examined how ERK5, that overlaps the functions of ERK1/2 in cell proliferation and survival, functions in a manner distinct from ERK1/2 in human AML cells induced to differentiate by 1,25D-dihydroxyvitamin D3 (1,25D). Using inhibitors of ERK1/2 and of MEK5/ERK5 at concentrations specific for each kinase in HL60 and U937 cells, we observed that selective inhibition of the kinase activity of ERK5, but not of ERK1/2, in the presence of 1,25D resulted in macrophage-like cell morphology and enhancement of phagocytic activity. Importantly, this was associated with increased expression of the macrophage colony stimulating factor receptor (M-CSFR), but was not seen when M-CSFR expression was knocked down. Interestingly, inhibition of ERK1/2 led to activation of ERK5 in these cells. Our results support the hypothesis that ERK5 negatively regulates the expression of M-CSFR, and thus has a restraining function on macrophage differentiation. The addition of pharmacological inhibitors of ERK5 may influence trials of differentiation therapy of AML. PMID:25447310

  9. The Arabidopsis Mediator Complex Subunit16 Positively Regulates Salicylate-Mediated Systemic Acquired Resistance and Jasmonate/Ethylene-Induced Defense Pathways[W

    PubMed Central

    Zhang, Xudong; Wang, Chenggang; Zhang, Yanping; Sun, Yijun; Mou, Zhonglin

    2012-01-01

    Systemic acquired resistance (SAR) is a long-lasting plant immunity against a broad spectrum of pathogens. Biological induction of SAR requires the signal molecule salicylic acid (SA) and involves profound transcriptional changes that are largely controlled by the transcription coactivator NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1). However, it is unclear how SAR signals are transduced from the NPR1 signaling node to the general transcription machinery. Here, we report that the Arabidopsis thaliana Mediator subunit16 (MED16) is an essential positive regulator of SAR. Mutations in MED16 reduced NPR1 protein levels and completely compromised biological induction of SAR. These mutations also significantly suppressed SA-induced defense responses, altered the transcriptional changes induced by the avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000/avrRpt2, and rendered plants susceptible to both Pst DC3000/avrRpt2 and Pst DC3000. In addition, mutations in MED16 blocked the induction of several jasmonic acid (JA)/ethylene (ET)–responsive genes and compromised resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. The Mediator complex acts as a bridge between specific transcriptional activators and the RNA polymerase II transcription machinery; therefore, our data suggest that MED16 may be a signaling component in the gap between the NPR1 signaling node and the general transcription machinery and may relay signals from both the SA and the JA/ET pathways. PMID:23064320

  10. Single and Combined Silencing of ERK1 and ERK2 Reveals Their Positive Contribution to Growth Signaling Depending on Their Expression Levels▿

    PubMed Central

    Lefloch, Renaud; Pouysségur, Jacques; Lenormand, Philippe

    2008-01-01

    The proteins ERK1 and ERK2 are highly similar, are ubiquitously expressed, and share activators and substrates; however, erk2 gene invalidation is lethal in mice, while erk1 inactivation is not. We ablated ERK1 and/or ERK2 by RNA interference and explored their relative roles in cell proliferation and immediate-early gene (IEG) expression. Reducing expression of either ERK1 or ERK2 lowered IEG induction by serum; however, silencing of only ERK2 slowed down cell proliferation. When both isoforms were silenced simultaneously, compensating activation of the residual pool of ERK1/2 masked a more deleterious effect on cell proliferation. It was only when ERK2 activation was clamped at a limiting level that we demonstrated the positive contribution of ERK1 to cell proliferation. We then established that ERK isoforms are activated indiscriminately and that their expression ratio correlated exactly with their activation ratio. Furthermore, we determined for the first time that ERK1 and ERK2 kinase activities are indistinguishable in vitro and that erk gene dosage is essential for survival of mice. We propose that the expression levels of ERK1 and ERK2 drive their apparent biological differences. Indeed, ERK1 is dispensable in some vertebrates, since it is absent from chicken and frog genomes despite being present in all mammals and fishes sequenced so far. PMID:17967895

  11. The sequence Pro295-Thr311 of the hinge region of oestrogen receptor α is involved in ERK1/2 activation via GPR30 in leiomyoma cells.

    PubMed

    Leiber, Denis; Burlina, Fabienne; Byrne, Cillian; Robin, Philippe; Piesse, Christophe; Gonzalez, Lucie; Leclercq, Guy; Tanfin, Zahra; Jacquot, Yves

    2015-11-15

    The ERα (oestrogen receptor α)-derived peptide ERα17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the Gαi protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of β-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ERα is down-regulated by prolonged treatment with E2 (oestradiol) or specific ERα siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ERα is required for the action of ERα17p. In addition, its PLM sequence, which interferes with the formation of the ERα-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ERα17p is, to our knowledge, the first known peptide targeting ERα-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects.

  12. Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways

    PubMed Central

    Yang, Lichao; Guo, Han; Li, Ying; Meng, Xianglan; Yan, Lu; Dan Zhang; Wu, Sangang; Zhou, Hao; Peng, Lu; Xie, Qiang; Jin, Xin

    2016-01-01

    The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 μg/ml) in the presence or absence of OEA (10, 20 and 40 μM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases. PMID:27721381

  13. The sequence Pro295-Thr311 of the hinge region of oestrogen receptor α is involved in ERK1/2 activation via GPR30 in leiomyoma cells.

    PubMed

    Leiber, Denis; Burlina, Fabienne; Byrne, Cillian; Robin, Philippe; Piesse, Christophe; Gonzalez, Lucie; Leclercq, Guy; Tanfin, Zahra; Jacquot, Yves

    2015-11-15

    The ERα (oestrogen receptor α)-derived peptide ERα17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the Gαi protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of β-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ERα is down-regulated by prolonged treatment with E2 (oestradiol) or specific ERα siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ERα is required for the action of ERα17p. In addition, its PLM sequence, which interferes with the formation of the ERα-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ERα17p is, to our knowledge, the first known peptide targeting ERα-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects. PMID:26371374

  14. Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases.

    PubMed

    Inamoto, Teruo; Azuma, Haruhito; Sakamoto, Takeshi; Kiyama, Satoshi; Ubai, Takanobu; Kotake, Yatsugu; Watanabe, Masahito; Katsuoka, Yoji

    2007-10-01

    Gamma-aminobutyric acid (GABA) was first discovered as an inhibitory neurotransmitter in the central nervous system (CNS) and has been reported to have a variety of functions, including regulation of cell division, cell differentiation and maturation, and to be involved in the development of certain cancers outside the CNS. In the present study, using the human renal cell carcinoma cell line Caki-2, we demonstrated that GABA stimulation significantly increased the expression of MMP-2 and -9 and subsequently increased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with GABA. It was found that GABA stimulation promoted the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. ERK1/2 phosphorylation was sustained for up to 12 h, while phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated GABA-induced MMP-9 expression and that both PD98059 and MMP inhibitors attenuated the GABA-induced invasive activity of Caki-2 cells. Moreover, data obtained by depletion of the MEK/ERK pathway using interfering RNA transfection of Caki-2 cells clearly corroborated the above results, as both MMP-9 expression and GABA-induced invasive ability were decreased significantly. We also demonstrated that the GABA-induced increase in invasive ability via ERK1/2 up-regulation was mediated mainly through the GABA-B receptor. These results indicate that GABA stimulation promotes cancer cell invasion and that the effect is partly due to ERK1/2-dependent up-regulation of MMPs.

  15. β-elemene inhibited expression of DNA methyltransferase 1 through activation of ERK1/2 and AMPKα signalling pathways in human lung cancer cells: the role of Sp1

    PubMed Central

    Zhao, ShunYu; Wu, Jingjing; Zheng, Fang; Tang, Qing; Yang, LiJun; Li, Liuning; Wu, WanYin; Hann, Swei Sunny

    2015-01-01

    β-elemene, a compound derived from Rhizoma zedoariae, is a promising new plant-derived drug with broad-spectrum anticancer activity. However, the underlying mechanism by which this agent inhibits human lung cancer cell growth has not been well elucidated. In this study, we showed that β-elemene inhibits human non-small cell lung carcinoma (NSCLC) cell growth, and increased phosphorylation of ERK1/2, Akt and AMPKα. Moreover, β-elemene inhibited expression of DNA methyltransferase 1 (DNMT1), which was not observed in the presence of the specific inhibitors of ERK (PD98059) or AMPK (compound C). Overexpression of DNMT1 reversed the effect of β-elemene on cell growth. Interestingly, metformin not only reversed the effect of β-elemene on phosphorylation of Akt but also strengthened the β-elemene-reduced DNMT1. In addition, β-elemene suppressed Sp1 protein expression, which was eliminated by either ERK1/2 or AMPK inhibitor. Conversely, overexpression of Sp1 antagonized the effect of β-elemene on DNMT1 protein expression and cell growth. Taken together, our results show that β-elemene inhibits NSCLC cell growth viaERK1/2- and AMPKα-mediated inhibition of transcription factor Sp1, followed by reduction in DNMT1 protein expression. Metformin augments the effect of β-elemene by blockade of Akt signalling and additively inhibition of DNMT1 protein expression. The reciprocal ERK1/2 and AMPKα signalling pathways contribute to the overall responses of β-elemene. This study reveals a potential novel mechanism by which β-elemene inhibits growth of NSCLC cells. PMID:25598321

  16. MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM

    PubMed Central

    Stojcheva, Nina; Schechtmann, Gennadi; Sass, Steffen; Roth, Patrick; Florea, Ana-Maria; Stefanski, Anja; Stühler, Kai; Wolter, Marietta; Müller, Nikola S.; Theis, Fabian J.; Weller, Michael; Reifenberger, Guido; Happold, Caroline

    2016-01-01

    Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo. PMID:26887050

  17. Over-expression of astrocytic ET-1 attenuates neuropathic pain by inhibition of ERK1/2 and Akt(s) via activation of ETA receptor.

    PubMed

    Hung, Victor K L; Tai, Lydia W; Qiu, Qiu; Luo, Xin; Wong, K L; Chung, Sookja K; Cheung, C W

    2014-05-01

    A differential role of endothelin-1 (ET-1) in pain processing has recently been suggested. However, the function of central ET-1 in neuropathic pain (NP) has not been fully elucidated to date. We report here the action of endogenous central ET-1 in sciatic nerve ligation-induced NP (SNL-NP) in a transgenic animal model that over-expresses ET-1 in the astrocytes (GET-1 mice). We hypothesized that the over-expression of astrocytic ET-1 would exert anti-allodynic and anti-hyperalgesic effects in NP, as demonstrated by mechanical threshold and plantar withdrawal latency using the von Frey filament and heat stimuli. In our animal model, GET-1 mice showed an increase in the withdrawal threshold and latency in response to the mechanical and thermal stimuli, respectively, in pain behavior tests after SNL. ET-1 and endothelin type A receptor (ETA-R) levels were increased significantly in L4-L6 segments of the spinal cord (ipsilateral to SNL) of GET-1 mice at 7 and 21days after surgery. Moreover, intrathecal administration of a specific ETA-R antagonist, BQ-123, attenuated the anti-allodynic and anti-hyperalgesic phenotype in GET-1 mice. The effects of BQ-123 on the mRNA expression of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase B/serine protein kinase (Akt(s)) were assessed in the ipsilateral L4-L6 segments harvested 30min after BQ-123 administration on day 7 after surgery. Phosphorylation of ERK1/2 and Akt(s) in the ipsilateral spinal cord of GET-1 mice was reduced following SNL, whereas no reduction was observed after intrathecal injection of BQ-123. In conclusion, our results showed that the xover-expression of astrocytic ET-1 reduced SNL-induced allodynia and hyperalgesia by inhibiting the activation of ERK1/2 and Akt(s) via the ETA-R-mediated pathway.

  18. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation.

    PubMed

    Murphy, Lynea A; Moore, Tanya; Nesnow, Stephen

    2012-04-15

    fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice.

  19. Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

    PubMed

    Galperin, Emilia; Abdelmoti, Lina; Sorkin, Alexander

    2012-01-01

    Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module. PMID:22606262

  20. A Form of Perforant Path LTP Can Occur without ERK1/2 Phosphorylation or Immediate Early Gene Induction

    ERIC Educational Resources Information Center

    Steward, Oswald; Huang, Fen; Guzowski, John F.

    2007-01-01

    Stimulation paradigms that induce perforant path long-term potentiation (LTP) initiate phosphorylation of ERK1/2 and induce expression of a variety of immediate early genes (IEGs). These events are thought to be critical components of the mechanism for establishing the changes in synaptic efficacy that endure for hours or longer. Here we show that…

  1. Injury-induced activation of ERK 1/2 in the sciatic nerve of healthy and diabetic rats.

    PubMed

    Stenberg, Lena; Kanje, Martin; Mårtensson, Lisa; Dahlin, Lars B

    2011-01-26

    Phosphorylation of extracellular-signal-regulated kinase 1/2 (p-ERK 1/2) was investigated by immunohistochemistry at 30 min, 1 h, and 48 h after nerve transection in the sciatic nerve of healthy and diabetic [streptozotocin (STZ)-induced diabetes mellitus and BioBreeding (BB; i.e. DR.lyp/lyp or BBDP)] rats. Transection injury increased the intensity of p-ERK 1/2 in nerve stumps at all time points. Staining was confined to Schwann cells with occasional faint staining in single axons. In diabetic rats, a lower intensity of p-ERK 1/2 was found at 1 and 48 h in the distal and proximal nerve stumps compared with healthy rats. STZ-induced diabetic rats were not different from BB rats. p-ERK 1/2 is activated differentially in Schwann cells after nerve injury in diabetic rats, whereas activation in STZ-induced diabetic rats did not differ from BB rats.

  2. CHANGES IN EXPRESSION OF PHOSPHORYLATED AND TOTAL ERK 1/2 IN TCDD-EXPOSED EMBRYONIC MOUSE PALATES

    EPA Science Inventory

    CHANGES IN EXPRESSION OF PHOSPHORYLATED AND TOTAL ERK1/2 IN TCDD-EXPOSED EMBRYONIC MOUSE PALATES.
    C Wolf and B Abbott, USEPA, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC 27711

    2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate...

  3. Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase.

    PubMed

    Shemetov, Anton A; Seit-Nebi, Alim S; Gusev, Nikolai B

    2011-09-01

    A number of phosphomimicking mutants (replacement of Ser/Thr residues by Asp) of human small heat shock protein HspB8 were obtained and phosphorylation of the wild type HspB8 and its mutants by ERK1 kinase was analyzed in vitro. Mutation S159D does not affect phosphorylation, whereas mutations S24D and S27D equally moderately inhibited and mutation T87D strongly inhibited phosphorylation of HspB8. The double mutations S24D/T87D and S27D/T87D induced very strong inhibitory effect and the triple mutations S24D/S27D/T87D completely prevented phosphorylation catalyzed by ERK1. Thus, Ser24 and Thr87, found to be phosphorylated in vivo, are among the sites phosphorylated by ERK1 in HspB8 in vitro. Mutations S24D and T87D affect intrinsic tryptophan fluorescence and susceptibility to chymotrypsinolysis of HspB8. Phosphomimicking mutations and phosphorylation promote concentration-dependent association of HspB8 subunits. Mutations S24D and S27D decrease, whereas mutation T87D increases the chaperone-like activity of HspB8. It is concluded that phosphorylation catalyzed by ERK1 might affect the structure and chaperone-like activity of HspB8 and therefore can be important for regulation of interaction of HspB8 with different target proteins.

  4. Fisetin suppresses ADAM9 expression and inhibits invasion of glioma cancer cells through increased phosphorylation of ERK1/2.

    PubMed

    Chen, Chien-Min; Hsieh, Yi-Hsien; Hwang, Jin-Ming; Jan, Hsun-Jin; Hsieh, Shu-Ching; Lin, Shin-Huey; Lai, Chung-Yu

    2015-05-01

    Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid which is widely distributed in plants. It has been reported to possess some anticancer and anti-invasive capabilities. We set out to explore the effects of fisetin on antimetastatic and its mechanism of action in GBM8401 cells. The results indicated that fisetin exhibited effective inhibition of cell migration and inhibited the invasion of GBM8401 cells under non-cytotoxic concentrations. To identify the potential targets of fisetin, human proteinase antibody array analysis was performed, and the results indicated that the fisetin treatment inhibited the expression of ADAM9 protein and mRNA, which are known to contribute to the progression of glioma cancer. Our results showed that fisetin phosphorylated ERK1/2 in a sustained way that contributed to the inhibited ADAM9 protein and mRNA expression determined by Western blot and RT-PCR. Moreover, inhibition of ERK1/2 by U0126 or transfection with the siERK plasmid significantly abolished the fisetin-inhibited migration and invasion through activation of the ERK1/2 pathway. In summary, our results suggest that fisetin might be a potential therapeutic agent against human glioma cells based on its capacity to activate ERK1/2 and to inhibit ADAM9 expression.

  5. Different activation of ERK1/2 and p38 with hyperbaric oxygen in dorsal root ganglion.

    PubMed

    Li, Kai-Cheng; Bao, Xiao-Chen; Fang, Yi-Qun; Ma, Jun; Meng, Miao; Chen, Hai-Ting; Li, Chi

    2011-01-01

    Prolonged hyperbaric oxygen exposure causes pulmonary and nervous system toxicity, although hyperbaric oxygen treatment has been used to treat a broad spectrum of ailments. In the current study, animals have been exposed to 100% oxygen at a pressure of 2.3 atmospheres absolute (ATA) for two, six and 10 hours or 0.23 MPa normoxic hyperbaric nitrogen (N2-O2 mixture, oxygen partial pressure = 21 kPa) for 10 hours. Then we investigated whether ERK1/2 and p38 had been activated in the dorsal root ganglion (DRG) by hyperbaric conditions. Using Western blot analysis, we found that the phosphorylation levels of ERK1/2 (phospho-ERK1/2) increased significantly (p < 0.05, n = 3 for each group) in the six-hour treatment of 100% oxygen at a pressure of 2.3 ATA. The phosphorylation levels of p38 (phospho-p38) increased significantly (p < 0.05, n = 3 for each group) in the 10-hour treatment of 100% oxygen at a pressure of 2.3 ATA--which was consistent with time course changes of an apoptosis marker, cleavage caspase-3--while the phospho-p38 decreased in the 10 hours of N2-O2 mixture. These results demonstrate that the ERK1/2 and p38 have been differently activated in the DRG by prolonged hyperbaric oxygen exposure.

  6. A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib.

    PubMed

    Sinnberg, Tobias; Makino, Elena; Krueger, Marcel A; Velic, Ana; Macek, Boris; Rothbauer, Ulrich; Groll, Nicola; Pötz, Oliver; Czemmel, Stefan; Niessner, Heike; Meier, Friedegund; Ikenberg, Kristian; Garbe, Claus; Schittek, Birgit

    2016-06-01

    Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that β-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that β-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, β-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of β-catenin in melanoma progression and therapy resistance. We further demonstrate that β-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi. PMID:27428425

  7. Acute liver failure impairs function and expression of breast cancer-resistant protein (BCRP) at rat blood-brain barrier partly via ammonia-ROS-ERK1/2 activation.

    PubMed

    Li, Ying; Zhang, Ji; Xu, Ping; Sun, Binbin; Zhong, Zeyu; Liu, Can; Ling, Zhaoli; Chen, Yang; Shu, Nan; Zhao, Kaijing; Liu, Li; Liu, Xiaodong

    2016-07-01

    We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300 mg/kg) for 2 days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5 mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly

  8. Acute liver failure impairs function and expression of breast cancer-resistant protein (BCRP) at rat blood-brain barrier partly via ammonia-ROS-ERK1/2 activation.

    PubMed

    Li, Ying; Zhang, Ji; Xu, Ping; Sun, Binbin; Zhong, Zeyu; Liu, Can; Ling, Zhaoli; Chen, Yang; Shu, Nan; Zhao, Kaijing; Liu, Li; Liu, Xiaodong

    2016-07-01

    We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300 mg/kg) for 2 days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5 mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly

  9. Stimulatory Effects of Coumestrol on Embryonic and Fetal Development Through AKT and ERK1/2 MAPK Signal Transduction.

    PubMed

    Lim, Whasun; Song, Gwonhwa

    2016-12-01

    Successful establishment of pregnancy is required for fetal-maternal interactions regulating implantation, embryonic development and placentation. A uterine environment with insufficient growth factors and nutrients increases the incidence of intrauterine growth restriction (IUGR) leading to an impaired uterine environment. In the present study, we demonstrated the effects of the phytoestrogen coumestrol on conceptus development in the pig that is regarded as an excellent biomedical animal model for research on IUGR. Results of this study indicated that coumestrol induced migration of porcine trophectoderm (pTr) cells in a concentration-dependent manner. In response to coumestrol, the phosphorylation of AKT, P70S6K, S6, ERK1/2 MAPK, and P90RSK proteins were activated in pTr cells and ERK1/2 MAPK and P90RSK phosphorylation was prolonged for a longer period than for the other proteins. To identify the signal transduction pathway induced by coumestrol, pharmacological inhibitors U0126 (an ERK1/2 inhibitor) and LY294002 (a PI3K inhibitor) were used to pretreat pTr cells. The results showed that coumestrol-induced phosphorylation of ERK1/2 MAPK and P90RSK was blocked by U0126. In addition, the increased phosphorylation in response to coumestrol was completely inhibited following pre-treatment incubation of pTr cells in the presence of LY294002 and U0126. Furthermore, these two inhibitors suppressed the ability of coumestrol to induce migration of pTr cells. Collectively, these findings suggest that coumestrol affects embryonic development through activation of the PI3K/AKT and ERK1/2 MAPK cell signal transduction pathways and improvement in the uterine environment through coumestrol supplementation may provide beneficial effects of enhancing embryonic and fetal survival and development. J. Cell. Physiol. 231: 2733-2740, 2016. © 2016 Wiley Periodicals, Inc.

  10. Chikusetsu saponin IVa confers cardioprotection via SIRT1/ERK1/2 and Homer1a pathway.

    PubMed

    Duan, Jialin; Yin, Ying; Wei, Guo; Cui, Jia; Zhang, Enhu; Guan, Yue; Yan, Jiajia; Guo, Chao; Zhu, Yanrong; Mu, Fei; Weng, Yan; Wang, Yanhua; Wu, Xiaoxiao; Xi, Miaomiao; Wen, Aidong

    2015-01-01

    Hyperglycemia-induced reactive oxygen species (ROS) generation and Ca(2+) overload contribute to the development of diabetic cardiomyopathy. In this study, we aimed to study the protective effects of Chikusetsu saponin IVa (CHS) from Aralia taibaiensis against hyperglycemia-induced myocardial injuries. Treatment of H9c2 cells with high glucose (HG) for 24 h resulted in a loss of cell viability and increase of ROS, LDH and Ca(2+) levels, and also induced cell apoptosis, and those changes were all markedly reversed by the administration of CHS. In further studies, CHS dose-dependently increased the expression of Homer1a, ERK1/2 and SIRT1 in both H9c2 cells and rat primary cardiomyocytes. However, transfection of Homer1a-specific siRNA abolished the ability of CHS in controlling the ROS and Ca(2+) homeostasis. Moreover, specific SIRT1 inhibitors or siRNA significantly suppressed the enhanced phosphorylation of ERK1/2 and expression of Homer1a induced by CHS as well as its cytoprotective effect. CHS induced Homer1a expression was also suppressed by siERK1/2. Additionally, results in diabetic mice also showed that CHS protected myocardium from I/R-introduced apoptosis by activating the SIRT1/ERK1/2/Homer1a pathway. These results demonstrated that CHS protected against hyperglycemia-induced myocardial injury through SIRT1/ERK1/2 and Homer1a pathway in vivo and in vitro. PMID:26648253

  11. Long noncoding RNA H19 controls DUSP5/ERK1/2 axis in cardiac fibroblast proliferation and fibrosis.

    PubMed

    Tao, Hui; Cao, Wei; Yang, Jing-Jing; Shi, Kai-Hu; Zhou, Xiao; Liu, Li-Ping; Li, Jun

    2016-01-01

    Down-regulation of DUSP5 has been shown to increase cell proliferation. DUSP5 expression is regulated through epigenetic events involving LncRNA H19 human choriocarcinoma cell line. However, the molecular mechanisms of H19 modulating the DUSP5 expression in cardiac fibrosis remain largely unknown. Here, we identify H19 negatively regulation of DUSP5 gene expression in cardiac fibroblast and fibrosis tissues. In vivo, the expression levels of H19, DUSP5, α-SMA, p-ERK1/2, and ERK1/2 in cardiac fibrosis tissue were estimated by Western blotting, quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. In vitro stimulation of freshly isolated rat cardiac fibroblasts with recombinant marine TGF-β1 was performed, followed by quantitative reverse transcription-polymerase chain reaction and Western blotting to detect changes in H19, DUSP5, p-ERK1/2, and ERK1/2 levels. Cardiac fibroblasts were transfected with pEX-3-H19 overexpressing, H19-RNAi down-regulating, or pEGFP-C1-DUSP5 overexpressing. Finally, cell proliferation was assessed by the MTT assay and cell cycle. H19 endogenous expression is overexpressed in cardiac fibroblast and fibrosis tissues, and an opposite pattern is observed for DUSP5. H19 ectopic overexpression reduces DUSP5 abundance and increases the proliferation of cardiac fibroblast, whereas H19 silencing causes the opposite effects. In a broader perspective, these results demonstrated that LncRNA H19 contributes to cardiac fibroblast proliferation and fibrosis, which act in part through repression of DUSP5/ERK1/2. PMID:27318893

  12. Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer

    PubMed Central

    van der Mijn, Johannes C.; Sol, Nik; Mellema, Wouter; Jimenez, Connie R.; Piersma, Sander R.; Dekker, Henk; Schutte, Lisette M.; Smit, Egbert F.; Broxterman, Henk J.; Skog, Johan; Tannous, Bakhos A.; Wurdinger, Thomas; Verheul, Henk M. W.

    2014-01-01

    Background Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005). Conclusion Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer. PMID:25491250

  13. Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases.

    PubMed

    Torres-Martínez, Marilu; Rubio-Infante, Néstor; García-Hernández, Ana Lilia; Nava-Acosta, Raúl; Ilhuicatzi-Alvarado, Damaris; Moreno-Fierros, Leticia

    2016-09-01

    The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways. PMID:27394658

  14. Ruta graveolens L. induces death of glioblastoma cells and neural progenitors, but not of neurons, via ERK 1/2 and AKT activation.

    PubMed

    Gentile, Maria Teresa; Ciniglia, Claudia; Reccia, Mafalda G; Volpicelli, Floriana; Gatti, Monica; Thellung, Stefano; Florio, Tullio; Melone, Mariarosa A B; Colucci-D'Amato, Luca

    2015-01-01

    Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens' effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue's noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.

  15. Ruta graveolens L. Induces Death of Glioblastoma Cells and Neural Progenitors, but Not of Neurons, via ERK 1/2 and AKT Activation

    PubMed Central

    Gentile, Maria Teresa; Volpicelli, Floriana; Gatti, Monica; Thellung, Stefano; Florio, Tullio; Melone, Mariarosa A. B.; Colucci-D’Amato, Luca

    2015-01-01

    Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens’ effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue’s noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention. PMID:25785932

  16. Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

    PubMed

    Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

    2014-01-15

    Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers. PMID:24291221

  17. Upregulation of cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on magnesium alloy surface coating with tricalcium phosphate.

    PubMed

    Jiang, Tianlong; Guo, Lei; Ni, Shenghui; Zhao, Yuyan

    2015-04-01

    Magnesium (Mg) alloys have been demonstrated to be viable orthopedic implants because of mechanical and biocompatible properties similar to natural bone. In order to improve its osteogenic properties, a porous β-tricalcium phosphate (β-TCP) was coated on the Mg-3AI-1Zn alloy by alkali-heat treatment technique. The human bone-derived cells (SaOS-2) were cultured on (β-TCP)-Mg-3AI-1Zn in vitro, and the osteoblast response, the morphology and the elements on this alloy surface were investigated. Also, the regulation of key intracellular signalling proteins was investigated in the SaOS-2 cells cultured on alloy surface. The results from scanning electron microscope and immunofluorescence staining demonstrated that (β-TCP)-Mg-3AI-1Zn induced significant osteogenesis. SaOS-2 cell proliferation was improved by β-TCP coating. Moreover, the (β-TCP)-Mg-3AI-1Zn surface induced activation of key intracellular signalling proteins in SaOS-2 cells. We observed an enhanced activation of Src homology and collagen (Shc), a common point of integration between bone morphogenetic protein 2, and the Ras/mitogen-activated protein kinase (MAPK) pathway. ERK1/2 MAP kinase activation was also upregulated, suggesting a role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the (β-TCP)-Mg-3AI-1Zn. These results suggest that β-TCP coating may contribute to successful osteoblast function on Mg alloy surface. (β-TCP)-Mg-3AI-1Zn may upregulate cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on Mg alloy surface.

  18. Spaced training rescues memory and ERK1/2 signaling in fragile X syndrome model mice.

    PubMed

    Seese, Ronald R; Wang, Kathleen; Yao, Yue Qin; Lynch, Gary; Gall, Christine M

    2014-11-25

    Recent studies have shown that short, spaced trains of afferent stimulation produce much greater long-term potentiation (LTP) than that obtained with a single, prolonged stimulation episode. The present studies demonstrate that spaced training regimens, based on these LTP timing rules, facilitate learning in wild-type (WT) mice and can offset learning and synaptic signaling impairments in the fragile X mental retardation 1 (Fmr1) knockout (KO) model of fragile X syndrome. We determined that 5 min of continuous training supports object location memory (OLM) in WT but not Fmr1 KO mice. However, the same amount of training distributed across three short trials, spaced by one hour, produced robust long-term memory in the KOs. At least three training trials were needed to realize the benefit of spacing, and intertrial intervals shorter or longer than 60 min were ineffective. Multiple short training trials also rescued novel object recognition in Fmr1 KOs. The spacing effect was surprisingly potent: just 1 min of OLM training, distributed across three trials, supported robust memory in both genotypes. Spacing also rescued training-induced activation of synaptic ERK1/2 in dorsal hippocampus of Fmr1 KO mice. These results show that a spaced training regimen designed to maximize synaptic potentiation facilitates recognition memory in WT mice and can offset synaptic signaling and memory impairments in a model of congenital intellectual disability.

  19. ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect

    PubMed Central

    Yang, Weiwei; Zheng, Yanhua; Xia, Yan; Ji, Haitao; Chen, Xiaomin; Guo, Fang; Lyssiotis, Costas A.; Aldape, Kenneth; Cantley, Lewis C.; Lu, Zhimin

    2012-01-01

    SUMMARY Pyruvate kinase M2 (PKM2) is upregulated in multiple cancer types and contributes to the Warburg effect by unclarified mechanisms. Here we demonstrate that EGFR-activated ERK2 binds directly to PKM2 I429/L431 via the ERK2 docking groove and phosphorylates PKM2 Ser37 but not PKM1. Phosphorylated PKM2 Ser37 recruits PIN1 for cis-trans isomerization of PKM2, which leads to PKM2 binding to importin α5 and nuclear translocation. Nuclear PKM2, acting as a coactivator of β-catenin, induces c-Myc expression, resulting in the upregulation of GLUT1, LDHA, and, in a positive feedback loop, PTB-dependent PKM2 expression. Replacement of wild type PKM2 with a nuclear translocation-deficient mutant (S37A) blocks the EGFR-promoted Warburg effect and brain tumor development. In addition, levels of PKM2 S37 phosphorylation correlate with EGFR and ERK1/2 activity in human glioblastoma specimens. Our findings highlight the importance of nuclear functions of PKM2 in the Warburg effect and tumorigenesis. PMID:23178880

  20. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    SciTech Connect

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen

    2012-04-15

    fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice. Highlights: ► Propiconazole increases cell proliferation in AML12 mouse hepatocytes. ► Propiconazole increases Ras farnesylation and alters Ras membrane localization. ► Propiconazole increases Erk1/2 phosphorylation. ► Dysregulation of the cholesterol biosynthesis pathway can explain these results. ► These results can explain similar effects induced by propiconazole in mice.

  1. 2-Arachidonylglyceryl ether and abnormal cannabidiol-induced vascular smooth muscle relaxation in rabbit pulmonary arteries via receptor-pertussis toxin sensitive G proteins-ERK1/2 signaling.

    PubMed

    Su, Judy Y; Vo, Anhkiet C

    2007-03-22

    The receptor(s) used by cannabinoids to relax vascular smooth muscle is unknown. Here, we investigated the effects of 2-arachidonylglyceryl ether (2-AG ether), a metabolically stable endocannabinoid, and abnormal cannabidiol (abn-CBD) on relaxation of permeabilized pulmonary arterial strips monitored with force, and on extracellular signal-regulated mitogen-activated protein kinases (ERK1/2) phosphorylation in permeabilized vascular smooth muscle cells using immunoblotting. We found that 2-AG ether and abn-CBD caused relaxation and increased phosphorylation of ERK1/2. 2-AG ether effects were completely abolished by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A), and partially blocked by (-)-1.3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol (O-1918). In contrast, abn-CBD effects were completely abolished by O-1918, and only partially blocked by AM251, and SR141716A. Both 2-AG ether and abn-CBD effects were partially blocked by pertussis toxin, an inhibitor of Gi/o proteins. PD98059, an inhibitor of mitogen activated protein kinase kinase (MEK), completely abolished the relaxation, but only partially blocked the increased phosphorylation of ERK1/2 by 2-AG ether. In contrast, abn-CBD-induced relaxation was partially blocked and the increased phosphorylation of ERK1/2 was abolished by PD98059. These findings suggest that 2-AG ether and abn-CBD-induced vascular smooth muscle relaxation are mediated by the cannabinoid CB1 receptor, and the abn-CBD receptor, respectively, and are modulated by cross-talk between the receptors. These responses occur mainly by coupling to pertussis toxin sensitive G proteins, but also, in part independent of these G proteins, which have been classically thought to initiate MEK/ERK1/2 signaling to relax vascular smooth muscle.

  2. Activation of villous trophoblastic p38 and ERK1/2 signaling pathways in preterm preeclampsia and HELLP syndrome.

    PubMed

    Szabo, Szilvia; Mody, Meera; Romero, Roberto; Xu, Yi; Karaszi, Katalin; Mihalik, Noemi; Xu, Zhonghui; Bhatti, Gaurav; Fule, Tibor; Hupuczi, Petronella; Krenacs, Tibor; Rigo, Janos; Tarca, Adi L; Hassan, Sonia S; Chaiworapongsa, Tinnakorn; Kovalszky, Ilona; Papp, Zoltan; Than, Nandor Gabor

    2015-07-01

    Preterm preeclampsia is associated with the failure of trophoblast invasion, placental hypoxic/ischemic injury and the release of toxic substances, which promote the terminal pathway of preeclampsia. In term preeclampsia, factors yet unknown trigger the placenta to induce the terminal pathway. The contribution of the villous trophoblast to these pathologic events has not been fully elucidated. Here we aimed to study how stress and signaling pathways influence trophoblastic functions in various subforms of preeclampsia. Tissue microarrays (TMAs) were constructed from placentas obtained from pregnant women in the following groups: 1-2) preterm preeclampsia with (n = 8) or without (n = 7) HELLP syndrome; 3) late-onset preeclampsia (n = 8); 4-5) preterm (n = 5) and term (n = 9) controls. TMA slides were stained for phosphorylated Akt-1, ERK1/2, JNK, and p38 kinases, and trophoblastic immunostainings were semi-quantitatively evaluated. BeWo cells were kept in various stress conditions, and the expression of FLT1, GCM1, LEP, and PGF was profiled by qRT-PCR, while Akt-1, ERK1/2, JNK, and p38 kinase activities were measured with phospho-kinase immunoassays. We found that: 1) Placental LEP and FLT1 expression was up-regulated in preterm preeclampsia with or without HELLP syndrome compared to controls; 2) Mean pp38 immunoscore was higher in preterm preeclampsia, especially in cases with HELLP syndrome, than in controls. 3) Mean pERK1/2 immunoscore was higher in preterm preeclampsia with HELLP syndrome than in controls. 4) In BeWo cells, ischemia up-regulated LEP expression, and it increased JNK and decreased ERK1/2 activity. 5) Hypoxia up-regulated FLT1 and down-regulated PGF expression, and it increased ERK1/2, JNK and p38 activity. 6) IL-1β treatment down-regulated PGF expression, and it increased JNK and p38 activity. 7) The p38 signaling pathway had the most impact on LEP, FLT1 and PGF expression. In conclusion, hypoxic and ischemic stress, along

  3. Optimizing an Intermittent Stretch Paradigm Using ERK1/2 Phosphorylation Results in Increased Collagen Synthesis in Engineered Ligaments

    PubMed Central

    Paxton, Jennifer Z.; Hagerty, Paul; Andrick, Jonathan J.

    2012-01-01

    Dynamic mechanical input is believed to play a critical role in the development of functional musculoskeletal tissues. To study this phenomenon, cyclic uniaxial mechanical stretch was applied to engineered ligaments using a custom-built bioreactor and the effects of different stretch frequency, amplitude, and duration were determined. Stretch acutely increased the phosphorylation of p38 (3.5±0.74-fold), S6K1 (3.9±0.19-fold), and ERK1/2 (2.45±0.32-fold). The phosphorylation of ERK1/2 was dependent on time, rather than on frequency or amplitude, within these constructs. ERK1/2 phosphorylation was similar following stretch at frequencies from 0.1 to 1 Hz and amplitudes from 2.5% to 15%, whereas phosphorylation reached maximal levels at 10 min of stretch and returned toward basal within 60 min of stretch. Following a single 10-min bout of cyclic stretch, the cells remained refractory to a second stretch for up to 6 h. Using the phosphorylation of ERK1/2 as a guide, the optimum stretch paradigm was hypothesized to be 10 min of stretch at 2.5% of resting length repeated every 6 h. Consistent with this hypothesis, 7 days of stretch using this optimized intermittent stretch program increased the collagen content of the grafts more than a continuous stretch program (CTL=3.1%±0.44%; CONT=4.8%±0.30%; and INT=5.9%±0.56%). These results suggest that short infrequent bouts of loading are optimal for improving engineered tendon and ligament physiology. PMID:21902469

  4. Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

    PubMed

    Yamada, Aya; Futagi, Masaharu; Fukumoto, Emiko; Saito, Kan; Yoshizaki, Keigo; Ishikawa, Masaki; Arakaki, Makiko; Hino, Ryoko; Sugawara, Yu; Ishikawa, Momoko; Naruse, Masahiro; Miyazaki, Kanako; Nakamura, Takashi; Fukumoto, Satoshi

    2016-01-01

    Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

  5. Curcumin treatment alters ERK-1/2 signaling in vitro and inhibits nasopharyngeal carcinoma proliferation in mouse xenografts.

    PubMed

    Xie, Yi-Qiang; Wu, Xian-Bo; Tang, Song-Qi

    2014-01-01

    Curcumin, a plant phenol, has been used for centuries in traditional medicines for its anti-inflammatory and anti-neoplastic properties. The compound is believed to act on a range of proteins involved in cell cycle regulation. In this study, the effect of curcumin on ERK-1/2 pathway protein expression and on proliferation of nasopharyngeal carcinoma cells was investigated. CNE-2Z nasopharyngeal carcinoma cells were cultured with 10, 20, 40, or 80 μM curcumin for 24 h before proliferation was assessed by MTT colorimetry. Cell proliferation was increasingly inhibited as the concentration of curcumin increased (P<0.005). Additionally, Western blotting revealed that expression of p-ERK-1/2, MMP-9, and TIMP-1 was altered following curcumin treatment, also in a dose-dependent manner. Expression of p-ERK-1/2 and MMP-9 decreased, while expression of TIMP-1 increased (P<0.05). Finally, CNE-2Z cells were xenografted under the skin of 18 nude mice. Mice were treated with vehicle only (control), 24 mg/kg curcumin (low-dose group), or 50 mg/kg curcumin (high-dose group) every other day for 40 days beginning 24 h after xenografting. Compared to tumors from the control group, the volume and weight of xenograft tumors was significantly lower in both curcumin groups, with a higher magnitude of difference in the high-dose curcumin group (P<0.05). These results indicate that curcumin treatment can inhibit proliferation of nasopharyngeal carcinoma cells and alter expression of proteins in the ERK-1/2 signaling pathway. Therefore, curcumin warrants further investigation as a potential treatment for nasopharyngeal cancer.

  6. Gestational marginal zinc deficiency impaired fetal neural progenitor cell proliferation by disrupting the ERK1/2 signaling pathway.

    PubMed

    Nuttall, Johnathan R; Supasai, Suangsuda; Kha, Jennifer; Vaeth, Brandon M; Mackenzie, Gerardo G; Adamo, Ana M; Oteiza, Patricia I

    2015-11-01

    This study investigated if a marginal zinc deficiency during gestation in rats could affect fetal neural progenitor cell (NPC) proliferation through a down-regulation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. Rats were fed a marginally zinc-deficient or adequate diet from the beginning of gestation until embryonic day (E)19. The proportion of proliferating cells in the E19 fetal ventricular zone was decreased by marginal zinc deficiency. Immunostaining for phosphorylated ERK1/2 in the cerebral cortex was decreased in the marginal zinc fetuses, and this effect was strongest in the ventricular zone. Furthermore, phosphorylation of the upstream mitogen-activated ERK kinases (MEK1/2) was not affected, suggesting that marginal zinc deficiency could have increased ERK-directed phosphatase activity. Similar findings were observed in cultured rat embryonic cortical neurons and in IMR-32 neuroblastoma cells, in which zinc-deficiency decreased ERK1/2 phosphorylation without affecting MEK1/2 phosphorylation. Indeed, zinc deficiency increased the activity of the ERK-directed phosphatase protein phosphatase 2A (PP2A) in the fetal cortex and IMR-32 cells. Inhibition of PP2A with okadaic acid prevented the decrease in ERK phosphorylation and proliferation of zinc-deficient IMR-32 cells. Together these results demonstrated that decreased zinc availability reduces ERK1/2 signaling and decreased NPC proliferation as a consequence of PP2A activation. Disruption of fetal neurogenesis could underlie irreversible neurobehavioral impairments observed after even marginal zinc nutrition during a critical period of early brain development.

  7. Prolactin-Stimulated Activation of ERK1/2 Mitogen-Activated Protein Kinases is Controlled by PI3-Kinase/Rac/PAK Signaling Pathway in Breast Cancer Cells

    PubMed Central

    Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; Kholodenko, Boris N.; Hoek, Jan B.; Kiyatkin, Anatoly

    2011-01-01

    There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells. PMID:21726627

  8. Induction of neuritogenesis in PC12 cells by a pulsed electromagnetic field via MEK-ERK1/2 signaling.

    PubMed

    Kudo, Tada-aki; Kanetaka, Hiroyasu; Shimizu, Yoshinaka; Abe, Toshihiko; Mori, Hitoshi; Mori, Kazumi; Suzuki, Eizaburo; Takagi, Toshiyuki; Izumi, Shin-ichi

    2013-01-01

    We examined the regulation of neuritogenesis by a pulsed electromagnetic field (PEMF) in rat PC12 pheochromocytoma cells, which can be induced to differentiate into neuron-like cells with elongated neurites by inducers such as nerve growth factor (NGF). Plated PC12 cells were exposed to a single PEMF (central magnetic flux density, 700 mT; frequency, 0.172 Hz) for up to 12 h per day and were then evaluated for extent of neuritogenesis or acetylcholine esterase (AChE) activity. To analyze the mechanism underlying the effect of the PEMF on the cells, its effects on intracellular signaling were examined using the ERK kinase (MEK) inhibitors PD098059 and U0126 (U0124 was used as a negative control for U0126). The number of neurite-bearing PC12 cells and AChE activity increased after PEMF exposure without the addition of other inducers of neuritogenesis. Additionally, PEMF exposure induced sustained activation of ERK1/2 in PC12 cells, but not in NR8383 rat alveolar macrophages. Furthermore, U0126 strongly inhibited PEMF-dependent ERK1/2 activation and neuritogenesis. The PEMF-dependent neuritogenesis was also suppressed by PD098059, but not U0124. These results suggest that PEMF stimulation independently induced neuritogenesis and that activation of MEK-ERK1/2 signaling was induced by a cell-type-dependent mechanism required for PEMF-dependent neuritogenesis in PC12 cells.

  9. Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

    PubMed Central

    Jin, Xiao-lu; Li, Peng-fei; Zhang, Chun-bing; Wu, Jin-ping; Feng, Xi-lian; Zhang, Ying; Shen, Mei-hong

    2016-01-01

    Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1–3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA. PMID:27630691

  10. Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway.

    PubMed

    Jin, Xiao-Lu; Li, Peng-Fei; Zhang, Chun-Bing; Wu, Jin-Ping; Feng, Xi-Lian; Zhang, Ying; Shen, Mei-Hong

    2016-07-01

    Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA. PMID:27630691

  11. Effects of neonatal corticosterone and environmental enrichment on retinal ERK1/2 and CREB phosphorylation in adult mice.

    PubMed

    Matteucci, Andrea; Ceci, Chiara; Mallozzi, Cinzia; Macrì, Simone; Malchiodi-Albedi, Fiorella; Laviola, Giovanni

    2014-11-01

    Exposure to Stimulating Environments (SE) during development may improve neuroplasticity in central nervous system, protect against neurotoxic damage, and promote neuronal recovery in adult life. While biochemical mechanisms of SE-promoted neuronal plasticity are well known in the brain, much less is known on the signaling cascade governing plasticity and neuroprotection in the retina. In order to investigate if in the retina signaling molecules involved in neuronal plasticity are affected by SE, neonatal CD-1 mice were exposed to moderate corticosterone levels (NC), supplemented through maternal milk during the first postnatal week, or to environmental enrichment (EE) conditions (physical and social stimuli) from early adolescence. Our results showed that both NC and EE increased the phosphorylation level of Extracellularly Regulated Kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) in the adult retinal tissue. Furthermore, we observed that activated ERK1/2 was restricted to Müller cells, while pCREB was mostly present in the nuclei of retinal neurons. Neither NC, nor EE modified the expression of GFAP, a marker of Müller cells activation. In conclusion our results indicate that both NC and EE activate ERK1/2 and CREB in the retina and provide a biochemical background for the neuroprotective activity exerted by SE against retinal damage. Furthermore, they support the role of Müller glia as a key cell determinant of retinal neuroplasticity.

  12. Pinocembrin attenuates MPP(+)-induced neurotoxicity by the induction of heme oxygenase-1 through ERK1/2 pathway.

    PubMed

    Wang, Hongquan; Wang, Yumin; Zhao, Linan; Cui, Qifu; Wang, Yuehua; Du, Guanhua

    2016-01-26

    Our recent study demonstrated that pinocembrin (PB), the most abundant flavonoid in propolis, has neuroprotective effect against 1-methyl-4-phenylpyridinium (MPP(+))-induced SH-SY5Y neurotoxicity. However, the mechanism as how PB can induce neuroprotection is not known. In the present study, we demonstrate here that PB increased heme oxygenase-1 (HO-1) expression, which conferred protection against MPP(+)-induced cytotoxicity, because the inhibitor of HO-1 zinc protoporphyrin-IX attenuated the neuroprotection of PB. PB induced the phosphorylation of ERK1/2, and its cytoprotective effect was abolished by ERK1/2 inhibitors. Meanwhile, we have shown that MPP(+) induce the expression in a concentration-dependent manner in SH-SY5Y cells, which was further enhanced by PB. Taken together, the results suggest that PB enhances HO-1 expression to suppress MPP(+)-induced oxidative damage via ERK1/2 signaling pathways. These results revealed the mechanisms of PB enhances HO-1 expression, and contribute to shed some light on the mechanisms whereby PB inhibits the MPP(+)-induced neurotoxicity. These data indicated that PB might provide a valuable therapeutic strategy for the treatment of PD. PMID:26655464

  13. Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

    PubMed Central

    Jin, Xiao-lu; Li, Peng-fei; Zhang, Chun-bing; Wu, Jin-ping; Feng, Xi-lian; Zhang, Ying; Shen, Mei-hong

    2016-01-01

    Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1–3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

  14. The downregulation of OPN inhibits proliferation and migration and regulate activation of Erk1/2 in ECA-109 cells

    PubMed Central

    Xu, Song-Tao; Zou, Fa-Zhang; Cai, Li-Na; Xu, Wan-Ling

    2015-01-01

    Osteopontin (OPN) involves in tumor formation, and strongly correlated with the tumor progression. It was overexpressed in human esophageal squamous cell carcinoma (ESCC). To study the molecular mechanisms of OPN in ESCC, we examined its roles in inhibiting proliferation and invasion of ECA-109 (esophageal squamous cell carcinoma) cells. The expression of OPN gene was knockdown by RNA interference (RNAi) in the Eca-109 cell. The transcription level of OPN was to detect by reverse transcription-quantitative PCR (RT-qPCR). Western blot assay was performed to detect the expression of OPN, Caspase-3,Caspase-8, Caspase-9, ERK1/2, phospho-ERK1/2 and MMP2 after RNAi. The cell proliferation and apoptosis were detected by MTT and Hoechst33342 assay. Transwell inserts was used for detecting ECA-109 cell’s migration ability. The results shown that the level of OPN mRNA and protein was significantly reduced after RNAi. Proliferation and migration of cell line (ECA-109) was significantly inhibited in vitro. The protein phosphorylation and activation of ERK1/2 in the OPN RNAi group reduced significantly than the negative control groups. In Conclusion, the proliferation and migration of human ESCC can be inhibited by RNAi-targeting OPN. OPN can promote the expression of MMP2 through the ERK signaling pathways. OPN could serve as a potential therapeutic target for human ESCC. PMID:26131112

  15. TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways

    PubMed Central

    Cheng, Jung-Chien; Chang, Hsun-Ming; Fang, Lanlan; Sun, Ying-Pu; Leung, Peter C. K.

    2015-01-01

    Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells. PMID:25955392

  16. Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK

    PubMed Central

    Lv, Shuang; Xu, Qing-Yuan; Sun, En-Cheng; Zhang, Ji-Kai; Wu, Dong-Lai

    2016-01-01

    Bluetongue virus (BTV), a complex double-stranded segmented RNA virus, has been found to initiate cellular autophagy for its own benefit. Here, with a view to understanding the underlying mechanisms, we first systematically dissected the exact signaling network in BTV-induced autophagy. We found that the activity of mTOR, a crucial pivot, was inhibited by BTV1 infection, subsequently leading to downstream p70S6K suppression and autophagy initiation. We then explored the upstream regulators of mTOR and analyzed their activities via a series of assays. We found BTV1-induced autophagy to be independent of the ERK1/2 signaling pathway. However, the BTV1-induced inhibition of PI3K/Akt was found to be partially responsible for mTOR inactivation and subsequent autophagy initiation. Furthermore, we found unexpectedly that AMPK seemed to play a more important role in BTV1-induced autophagy. Elevated [Ca2+]cyto-mediated activation of CaMKKβ exactly managed the activation of AMPK, which then positively regulated autophagy through suppressing mTOR. We must emphasize that TSC2 is a fatal mediator between upstream Akt or AMPK and downstream mTOR through its phosphorylation. Taken together, our data suggested that the BTV1-induced inhibition of the Akt-TSC2-mTOR pathway and the upregulation of the AMPK-TSC2-mTOR pathway both contributed to autophagy initiation and further favored virus replication. PMID:26976147

  17. Activation of Transcription Factor GAX and Concomitant Downregulation of IL-1β and ERK1/2 Modulate Vascular Smooth Muscle Cell Phenotype in 3D Fibrous Scaffolds.

    PubMed

    Lin, Shigang; Mequanint, Kibret

    2015-09-01

    Since vascular smooth muscle cells (VSMCs) display phenotypic plasticity in response to changing environmental cues, understanding the molecular mechanisms underlying the phenotypic modulation mediated by a three-dimensional (3D) scaffold is important to engineer functional vasculature. Following cell seeding into 3D scaffolds, the synthetic phenotype is desired to enable cells to expand rapidly and produce and assemble extracellular matrix components, but must revert to a quiescent contractile phenotype after tissue fabrication to impart the contractile properties found in native blood vessels. This study shows that 3D electrospun fibrous scaffolds regulate human coronary artery smooth muscle cells (HCASMCs) toward a more synthetic phenotype characterized by reduced contractile markers, such as smooth muscle alpha-actin and calponin. The reduction in contractile markers expression was mediated by endogenously expressed proinflammatory cytokine interleukin-1β (IL-1β). 3D topography transiently induces concomitant upregulation of IL-1β and MAPK ERK1/2 through nuclear factor-κB-dependent signaling pathway. An early burst of expression of IL-1β is essential for suppression of the homeobox transcription factor Gax and related cyclin-dependent kinase inhibitor p21(Cip1), which are key regulators for cells exiting from cell cycle. Our findings provide new insights for understanding signaling mechanisms of HCASMCs in electrospun 3D fibrous scaffolds, which have considerable value for application in vascular tissue engineering. PMID:26041434

  18. TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways.

    PubMed

    Cheng, Jung-Chien; Chang, Hsun-Ming; Fang, Lanlan; Sun, Ying-Pu; Leung, Peter C K

    2015-01-01

    Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells.

  19. [MOLECULAR MECHANISMS OF ERK1/2 KINASES REGULATION IN THE GLUTAMATE- AND GABA-ERGIC NEURONS DURING SEIZURE EXPRESSION IN KRUSHINSKY-MOLODKINA RATS].

    PubMed

    Korotkov, A A; Glazova, M V; Nikitina, L S; Dorofeeva, N A; Kirillova, O D; Chernigovskaya, E V

    2015-10-01

    The aim of the present study was to analyze a role of the ERK1/2 signaling pathway in the regulation of excitation and inhibitory neurons in the hippocampus and the temporal cortex of Krushinsky-Molodkina rats during seizure development finalizing with ataxia. Analysis was done by Western bloting as well as by immunohistochemistry. The results demonstrated significant up-regulation of ERK1/2 activity in the hippocampus in several seconds after sound stimulation. At the same time increased ERK1/2 activity was correlated with enhanced level of SNARE protein SNAP-25 and activation of synapsin I, the proteins which regulate exocytosis machinery. Decreased level of VGLUT2 associated with activation of ERK1/2 and exocytosis proteins supposed activation of glutamate release in the hippocampus, while in the temporal cortex diminished activity of ERK1/2 and synapsin I associated with VGLUT2 up-regulation assumed inhibition of glutamatergic transmission. Our data let us supposed that decreasing of glutamate release in th& temporal cortex could be a trigger for the inhibition of hippocampal glutamatergic system and the beginning of further ataxia stage. Our data demonstrated correlation between expression and activity of exocytosis proteins and ERK1/2 mainly in the glutamategic neurons of the hippocampus and the temporal cortex that let us proposed significant role of ERK1/2 kinases as a positive regulator of glutamate release and as a result initiation of seizure expression. PMID:26827493

  20. High sodium augments angiotensin II-induced vascular smooth muscle cell proliferation through the ERK 1/2-dependent pathway.

    PubMed

    Liu, Gang; Hitomi, Hirofumi; Rahman, Asadur; Nakano, Daisuke; Mori, Hirohito; Masaki, Tsutomu; Ma, Hong; Iwamoto, Takahiro; Kobori, Hiroyuki; Nishiyama, Akira

    2014-01-01

    Angiotensin II (Ang II)-induced vascular injury is exacerbated by high-salt diets. This study examined the effects of high-sodium level on Ang II-induced cell proliferation in rat vascular smooth muscle cells (VSMCs). The cells were cultured in a standard medium containing 137.5 mmol l(-1) of sodium. The high-sodium medium (140 mmol l(-1)) contained additional sodium chloride. Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by western blot analysis. Cell proliferation was evaluated by [(3)H]-thymidine incorporation. Ang II (100 nmol l(-1)) significantly increased ERK 1/2 phosphorylation and cell proliferation in the both medium containing standard sodium and high sodium. High-sodium level augmented Ang II-induced ERK 1/2 phosphorylation and cell proliferation compared with standard sodium. Pre-treatment with candesartan (1 μmol l(-1), Ang II type 1 receptor blocker) or PD98095 (10 μmol l(-1), ERK kinase iinhibitor) abolished the proliferative effect induced by high sodium/Ang II. Pre-treatment with 5-N,N-hexamethylene amiloride (30 μmol l(-1), Na(+)/H(+) exchanger type 1 (NHE-1) inhibitor), but not SN-6 (10 μmol l(-1), Na(+)/Ca(2+) exchanger inhibitor) or ouabain (1 mmol l(-1), Na(+)/K(+)-ATPase inhibitor) attenuated ERK 1/2 phosphorylation or cell proliferation. Osmotic pressure or chloride had no effect on Ang II-induced proliferative changes. High-sodium level did not affect Ang II receptor expression. Ang II increased intracellular pH via NHE-1 activation, and high-sodium level augmented the pH increase induced by Ang II. These data suggest that high-sodium level directly augments Ang II-induced VSMC proliferation through NHE-1- and ERK 1/2-dependent pathways and may offer new insights into the mechanisms of vascular remodeling by high-sodium/Ang II.

  1. Learning to be different: Acquired skills, social learning, frequency dependence, and environmental variation can cause behaviourally mediated foraging specializations

    USGS Publications Warehouse

    Tinker, M.T.; Mangel, M.; Estes, J.A.

    2009-01-01

    Question: How does the ability to improve foraging skills by learning, and to transfer that learned knowledge, affect the development of intra-population foraging specializations? Features of the model: We use both a state-dependent life-history model implemented by stochastic dynamic programming (SDPM) and an individual-based model (IBM) to capture the dynamic nature of behavioural preferences in feeding. Variables in the SDPM include energy reserves, skill levels, energy and handling time per single prey item, metabolic rate, the rates at which skills are learned and forgotten, the effect of skills on handling time, and the relationship between energy reserves and fitness. Additional variables in the IBM include the probability of successful weaning, the logistic dynamics of the prey species with stochastic recruitment, the intensity of top-down control of prey by predators, the mean and variance in skill levels of new recruits, and the extent to which learned Information can be transmitted via matrilineal social learning. Key range of variables: We explore the effects of approaching the time horizon in the SDPM, changing the extent to which skills can improve with experience, increasing the rates of learning or forgetting of skills, changing whether the learning curve is constant, accelerating (T-shaped) or decelerating ('r'-shaped), changing both mean and maximum possible energy reserves, changing metabolic costs of foraging, and changing the rate of encounter with prey. Conclusions: The model results show that the following factors increase the degree of prey specialization observed in a predator population: (1) Experience handling a prey type can substantially improve foraging skills for that prey. (2) There is limited ability to retain complex learned skills for multiple prey types. (3) The learning curve for acquiring new foraging skills is accelerating, or J-shaped. (4) The metabolic costs of foraging are high relative to available energy reserves. (5

  2. The Internalization of Neurotensin by the Low-Affinity Neurotensin Receptors (NTSR2 and vNTSR2) Activates ERK 1/2 in Glioma Cells and Allows Neurotensin-Polyplex Transfection of tGAS1.

    PubMed

    Ayala-Sarmiento, Alberto E; Martinez-Fong, Daniel; Segovia, José

    2015-08-01

    Glioblastoma is the most malignant primary brain tumor and is very resistant to treatment; hence, it has a poor prognosis. Neurotensin receptor type 1 (NTSR1) plays a key role in cancer malignancy and has potential therapeutic applications. However, the presence and function of neurotensin (NTS) receptors in glioblastoma is not clearly established. RT-PCR assays showed that healthy (non-tumor) astroglial cells and C6 glioma cells express NTSR2 and its isoform (vNTSR2) rather than NTSR1. In glioma cells, NTS promotes the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2), an effect that was completely abolished by blocking the internalization of the NTS/NTSR complex. We demonstrated pharmacologically that the internalization is dependent on the activation of NTSR2 receptors and it was prevented by levocabastine, a NTSR2 receptor antagonist. The internalization of NTSR2 and vNTSR2 was further demonstrated by its ability to mediate gene transfer (transfection) via the NTS-polyplex system. Expression of reporter transgenes and of the pro-apoptotic soluble form of growth arrest specific 1 (tGAS1) was observed in glioma cells. A significant reduction on the viability of C6 cells was determined when tGAS1 was transfected into glioma cells. Conversely, astroglial cells could neither internalize NTS nor activate ERK 1/2 and could not be transfected by the NTS-polyplex. These results demonstrate that the internalization process of NTSR2 receptors is a key regulator necessary to trigger the activation of the ERK 1/2. Our data support a new internalization pathway in glioma C6 cells that involve NTSR2/vNTSR2, which can be used to selectively transfer therapeutic genes using the NTS-polyplex system.

  3. Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

    SciTech Connect

    Arai, Roberto J.; Debbas, Victor; Stern, Arnold; Monteiro, Hugo P.

    2008-12-01

    Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

  4. Major contribution of MEK1 to the activation of ERK1/ERK2 and to the growth of LS174T colon carcinoma cells

    SciTech Connect

    Shama, Jessica; Garcia-Medina, Raquel; Pouyssegur, Jacques Vial, Emmanuel

    2008-08-08

    Mammalian cells express two closely related MEK isoforms, MEK1 and MEK2, upstream of the ERK1/ERK2 MAPK module. Although genetic studies have suggested that MEK1 and MEK2 do not have overlapping functions in vivo, little is known about their specific contribution to the activation of ERKs and to tumor cell proliferation. We used Tet-inducible shRNA to investigate the independent role of MEK1 and MEK2 for the oncogenic and the serum-induced activation of ERK1 and ERK2 in LS174T colon carcinoma cells. We show that MEK1 is the main activator of both ERK1 and ERK2. MEK2 removal has no impact by itself but it can cooperate with MEK1 ablation for the inhibition of ERK1/2 activity. In addition, we show that MEK1 is the critical isoform regulating tumor cell proliferation in vitro and in vivo.

  5. Ghrelin promotes intestinal epithelial cell proliferation through PI3K/Akt pathway and EGFR trans-activation both converging to ERK 1/2 phosphorylation.

    PubMed

    Waseem, Talat; Duxbury, Mark; Ashley, Stanley W; Robinson, Malcolm K

    2014-02-01

    Little is known about ghrelin's effects on intestinal epithelial cells even though it is known to be a mitogen for a variety of other cell types. Because ghrelin is released in close proximity to the proliferative compartment of the intestinal tract, we hypothesized that ghrelin may have potent pro-proliferative effect on intestinal epithelial cells as well. To test this hypothesis, we characterized the effects of ghrelin on FHs74Int and Caco-2 intestinal epithelial cell lines in vitro. We found that ghrelin has potent dose dependent proliferative effects in both cell lines through a yet to be characterized G protein coupled growth hormone secretagogue receptor (GHS-R) subtype. Consistent with above findings, cell cycle flowcytometric analyses demonstrated that ghrelin shifts cells from the G1 to S phase and thereby promotes cell cycle progression. Further characterization of subcellular events, suggested that ghrelin mediates its pro-proliferative effect through Adenylate cyclase (AC)-independent epidermal growth factor receptor (EGFR) trans-activation and PI3K-Akt phosphorylation. Both these pathways converge to stimulate MAPK, ERK 1/2 downstream. The role of ghrelin in states where intestinal mucosal injury and rapid mucosal repair occur warrants further investigation.

  6. Targeting ERK1/2-bim signaling cascades by BH3-mimetic ABT-737 as an alternative therapeutic strategy for oral cancer.

    PubMed

    Shin, Ji-Ae; Kim, Lee-Han; Lee, Sook-Jeong; Jeong, Joseph H; Jung, Ji-Youn; Lee, Hae Nim; Hong, In-Sun; Cho, Sung-Dae

    2015-11-01

    To date, many different chemotherapeutic agents have been widely used as common treatments for oral cancers. However, their therapeutic effects have been disappointing, and these agents may have unwanted side effects. Among the many regulatory factors, overexpression of pro-survival Bcl-2 family members may promote resistance to chemotherapeutic drugs in many tumors. The BH3 domain-only proteins effectively antagonize their apoptotic activities. Therefore, there is substantial interest in developing chemotherapeutic drugs that directly target pro-survival Bcl-2 proteins by mimicking the BH3 domain and unleashing pro-apoptotic molecules in tumor cells. Among the numerous available small molecule BH3 mimetics, ABT-737, a potent small molecule that binds to Bcl-2/Bcl-xL with high affinity, has anti-tumor activity in a wide variety of cancer cells. However, the effects of ABT-737 on human oral cancers and the underlying molecular mechanisms have not previously been elucidated. In the present study, we observed that inactivation of the ERK1/2 signaling pathway using ABT-737 dramatically increased the expression of pro-apoptotic protein Bim via transcriptional and/or posttranslational regulation, in a cell type-dependent manner, inducing mitochondria-mediated apoptosis of human oral cancer cells. To the best of our knowledge, this is the first demonstration of the antitumor effects of ABT-737 on human oral cancers. PMID:26447615

  7. Selective β2-AR Blockage Suppresses Colorectal Cancer Growth Through Regulation of EGFR-Akt/ERK1/2 Signaling, G1-Phase Arrest, and Apoptosis.

    PubMed

    Chin, Chih-Chien; Li, Jhy-Ming; Lee, Kam-Fai; Huang, Yun-Ching; Wang, Kuan-Chieh; Lai, Hsiao-Ching; Cheng, Chih-Chung; Kuo, Yi-Hung; Shi, Chung-Sheng

    2016-02-01

    The stress-upregulated catecholamines-activated β1- and β2-adrenergic receptors (β1/2-ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of β1/2-ARs signaling for the treatment of CRC and elucidated the significance of β2-AR expression in CRC in vitro and in clinical samples. The impacts of β1/2-AR antagonists in CRC in vitro and CRC-xenograft in vivo were examined. We found that repression of β2-AR but not β1-AR signaling selectively suppressed cell viability, induced G1-phase cell cycle arrest, caused both intrinsic and extrinsic pathways-mediated apoptosis of specific CRC cells and inhibited CRC-xenograft growth in vivo. Moreover, the expression of β2-AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidence that the expression profiles, signaling, and blockage of β2-AR have a unique pattern in CRC comparing to other cancers. β2-AR antagonism selectively suppresses the growth of CRC accompanying active β2-AR signaling, which potentially carries wild-type KRAS, in vitro and in vivo via the inhibition of β2-AR transactivated EFGR-Akt/ERK1/2 signaling pathway. Thus, β2-AR blockage might be a potential therapeutic strategy for combating the progressions of β2-AR-dependent CRC.

  8. ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma-lamin A complexes.

    PubMed

    Rodríguez, Javier; Calvo, Fernando; González, José M; Casar, Berta; Andrés, Vicente; Crespo, Piero

    2010-11-29

    As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation.

  9. TGF-β1 promotes bovine mammary fibroblast proliferation through the ERK 1/2 signalling pathway.

    PubMed

    Gao, Yuanyuan; Wang, Yuping; Li, Yingying; Xia, Xiaojing; Zhao, Shuang; Che, Yanyi; Sun, Yingying; Lei, Liancheng

    2016-07-01

    The abnormal proliferation of bovine mammary fibroblasts (BMFBs) impairs mammary gland development and lactation. Severe manifestations develop into breast fibrosis, leading to the culling of cows and causing serious losses to the dairy industry. Transforming growth factor β1 (TGF-β1) is an important modulator of cell proliferation and extracellular matrix formation; however, limited information is available on BMFBs. In this study, a convenient and stable culture method for BMFBs was established. Treatment with 5 ng/mL of TGF-β1 significantly promoted the proliferation of BMFBs and accelerated the cell cycle. TGF-β1 stimulation for up to 12 h significantly increased the relative ERK1/2 mRNA expression and enhanced the protein expression of p-ERK1/2 and cyclin D1. Conversely, the ERK1/2 inhibitor PD98059 blocked these TGF-β1 effects. Further exploration using a mouse model showed that TGF-β1 significantly increased the proportion of fibroblasts and accelerating the cell transition from the G1 to G2/M phases. In addition, TGF-β1 enhanced the expression of fibrosis markers, α-SMA and I Collagen, which could be blocked efficiently by the PD98059 in mouse mammary gland. Finally, immunofluorescence analysis confirmed that TGF-β1 promoted fibroblast proliferation in healthy dairy cows after normal long-term dietary corn straw roughage supplementation. It is suggested that the diet may promote mammary fibroblast proliferation by raising the level of TGF-β1. Our study provides new insights into how nutrition causes undesirable changes in mammary gland structure.

  10. Regulation of involucrin in psoriatic epidermal keratinocytes: the roles of ERK1/2 and GSK-3β.

    PubMed

    Chen, Jia-Qi; Man, Xiao-Yong; Li, Wei; Zhou, Jiong; Landeck, Lilla; Cai, Sui-Qing; Zheng, Min

    2013-07-01

    Psoriasis, a common inflammatory skin disease, is characterized by epidermal hyperplasia, abnormal differentiation, angiogenesis, immune activation, and inflammation. Involucrin is an early terminal differentiation marker of epidermal keratinocytes. In this study, we determined the immunolocalization of involucrin in psoriatic lesions and normal skin of individuals without psoriasis by means of immunofluorescence (IF) assay. Furthermore, the regulation of involucrin by interleukin (IL)-13, IL-17A, endothelin (ET)-1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was investigated by Western blot. Extracellular regulate protein kinases 1/2 (ERK1/2) and glycogen syntheses kinase-3β (GSK-3β) inhibitors were also included to define the roles of these signals in the production of involucrin in both psoriatic and normal keratinocytes. In psoriatic lesional skin, involucrin was detected in the stratum spinosum, but not in the basal or the cornified layer. In normal skin, involucrin was restricted to the granular layer and the upper stratum spinosum. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ up-regulate expression of involucrin in both psoriatic and normal keratinocytes. However, this effect was abolished by ERK1/2 and GSK-3β inhibitors. In conclusion, involucrin is up-regulated in psoriatic keratinocytes. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ could increase involucrin protein levels in psoriatic and normal keratinocytes. The ERK1/2 and GSK-3β signaling pathways may play positive roles in regulating epidermal differentiation as observed in psoriasis.

  11. MicroRNA-203 negatively regulates c-Abl, ERK1/2 phosphorylation, and proliferation in smooth muscle cells.

    PubMed

    Liao, Guoning; Panettieri, Reynold A; Tang, Dale D

    2015-09-01

    The nonreceptor tyrosine kinase c-Abl has a role in regulating smooth muscle cell proliferation, which contributes to the development of airway remodeling in chronic asthma. MicroRNAs (miRs) are small noncoding RNA molecules that regulate gene expression by binding to complementary sequences in the 3' untranslated regions (3' UTR) of target mRNAs. Previous analysis suggests that miR-203 is able to bind to the 3' UTR of human c-Abl mRNA. In this report, treatment with miR-203 attenuated the expression of c-Abl mRNA and protein in human airway smooth muscle (HASM) cells. Furthermore, transfection with an miR-203 inhibitor enhanced the expression of c-Abl at mRNA and protein levels in HASM cells. Treatment with platelet-derived growth factor (PDGF) induced the proliferation and ERK1/2 phosphorylation in HASM cells. Exposure to miR-203 attenuated the PDGF-stimulated proliferation and ERK1/2 phosphorylation in HASM cells. The expression of c-Abl at protein and mRNA levels was higher in asthmatic HASM cells, whereas the level of miR-203 was reduced in asthmatic HASM cells as compared to control HASM cells. Taken together, our present results suggest that miR-203 is a negative regulator of c-Abl expression in smooth muscle cells. miR-203 regulates smooth muscle cell proliferation by controlling c-Abl expression, which in turn modulates the activation of ERK1/2.

  12. TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation

    PubMed Central

    Funcke, Jan-Bernd; Zoller, Verena; El Hay, Muad Abd; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2015-01-01

    Upon obesity, adipose tissue is excessively expanded and characterized by pathologic processes like hypoxia, fibrosis, and inflammation. Death ligands belonging to the TNF superfamily such as TNF-α are important contributors to these derangements and exert a pronounced influence on the metabolic and cellular homeostasis of adipose tissue. Here, we sought to identify the effect of the death ligand TNF-related apoptosis-inducing ligand (TRAIL) on the adipose tissue precursor cell pool and therefore investigated its influence on preadipocyte proliferation. Treatment of human preadipocytes with TRAIL resulted in a time- and dose-dependent increase in proliferation (EC50 3.4 ng/ml) comparable to IGF-1. Although no apoptosis was observed, TRAIL triggered a rapid cleavage of caspase-8 and -3. Neither inhibition of caspase activity by zVAD.fmk (20 µM) nor ablation of caspase-8 expression by lentivirus-delivered small hairpin RNA (shRNA) abolished the proliferative response. TRAIL triggered a delayed and sustained activation of ERK1/2, leaving Akt, p38, JNK, and NF-κB unaffected. Importantly, inhibition of ERK1/2 activation by PD0325901 (300 nM) or AZD6244 (5 or 10 µM) completely abolished the proliferative response. We thus reveal a hitherto unknown function of TRAIL in regulating adipose tissue homeostasis by promoting the proliferation of tissue-resident precursor cells.—Funcke, J.-B., Zoller, V., Abd El Hay, M., Debatin, K.-M., Wabitsch, M., Fischer-Posovszky, P. TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation. PMID:25857555

  13. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M.

    PubMed

    Thress, Kenneth S; Paweletz, Cloud P; Felip, Enriqueta; Cho, Byoung Chul; Stetson, Daniel; Dougherty, Brian; Lai, Zhongwu; Markovets, Aleksandra; Vivancos, Ana; Kuang, Yanan; Ercan, Dalia; Matthews, Sarah E; Cantarini, Mireille; Barrett, J Carl; Jänne, Pasi A; Oxnard, Geoffrey R

    2015-06-01

    Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

  14. Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway.

    PubMed

    Petecchia, Loredana; Sabatini, Federica; Usai, Cesare; Caci, Emanuela; Varesio, Luigi; Rossi, Giovanni A

    2012-08-01

    Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-a, IL-4 and IFN-g to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogenactivated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-a (47±3%), IL-4 (43±1%) and INF-g (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm²)1000 in control cultures and in those exposed to TNF-a, IFN-g and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were

  15. PD 098059, an inhibitor of ERK1 activation, attenuates the in vivo invasiveness of head and neck squamous cell carcinoma

    PubMed Central

    Simon, C; Hicks, M J; Nemechek, A J; Mehta, R; Jr, B W O'Malley; Goepfert, H; Flaitz, C M; Boyd, D

    1999-01-01

    Increased mortality of patients with oral cancer largely reflects the local and regional spread of the disease. The invasiveness of these tumours requires hydrolases which are regulated through AP-1-dependent transcriptional mechanisms. Since the amount/activity of transcription factors bound to the AP-1 motif are regulated partly through the extracellular signal-regulated kinases (ERK1/ERK2), we determined the effect of PD 098059, an inhibitor of ERK1/ERK2 activation, on the in vivo invasiveness of a human squamous cell carcinoma cell line (UM-SCC-1) derived from the oral cavity. We utilized the floor of mouth musculature consisting of the mylohyoid, geniohyoid and genioglossus muscle (which are sequentially arranged), as a natural barrier to assess tumour spread in vivo in the nude mouse. Mice were inoculated with tumour cells superficial to the mylohyoid muscle. After 18 days, tumours were injected with either empty liposomes (control) or liposomes containing 5 μM PD 098059 and, after an additional 22 days, the jaws of mice examined histologically. Highly infiltrative tumours, which had penetrated the genioglossus muscle, were evident in 10/12 control mice. In contrast, in 9/12 mice in which the tumours were injected with PD 098059, tumours did not extend beyond the mylohyoid or geniohyoid muscles. Tumours penetrated bone nutrient canals in 7/12 control mice but in only 3/12 PD 098059-treated mice. Neurotropism, characteristic of aggressive oral squamous cell carcinoma, was evident in 6/12 control mice but was completely abolished (0/12 mice) in the PD 098059-treated mice. Using a staging system based on the muscle layer involved, neurotropism, as well as bone involvement, we found the inhibition of invasion to be statistically significant (P < 0.01). The reduced invasiveness of the PD 098059-liposome-treated oral cancers was associated with diminished 92-kDa type IV collagenase and ERK1/ERK2 activities but was not a consequence of a slower tumour growth rate

  16. Potentiation of arsenic-induced cytotoxicity by sulfur amino acid deprivation (SAAD) through activation of ERK1/2, p38 kinase and JNK1: the distinct role of JNK1 in SAAD-potentiated mercury toxicity.

    PubMed

    Son, M H; Kang, K W; Lee, C H; Kim, S G

    2001-04-01

    Sulfur amino acid deficiency occurs in certain pathophysiological situations (e.g. protein-calorie malnutrition). Previous studies revealed that sulfur amino acid deprivation (SAAD) activated MAP kinases and potentiated cadmium-induced cytotoxicity by activation of ERK1/2 in conjunction with p38 kinase or JNK. The present study was designed to determine susceptibility of cells to a variety of heavy metals in combination with SAAD. Viability was assessed in H4IIE cells treated with sodium arsenite, mercuric chloride, sodium selenite, lead acetate, chromium trioxide or manganese chloride. SAAD potentiated the cytotoxicity of H4IIE cells by arsenic or mercury (i.e. EC50, 19 and 5 microM in SAAD vs. 401 and 42 microM in control medium, respectively). TUNEL assays revealed that the potentiated arsenic or mercury toxicity involved apoptotic cell death. Lead or selenite moderately elicited cell death, which was not enhanced by SAAD. Chromium or manganese caused no significant cytotoxicity. Treatment of cells with U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] an ERK1/2 inhibitor or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] a p38 kinase inhibitor effectively prevented SAAD-potentiated arsenic toxicity. The potentiated arsenic toxicity was also inhibited in cells stably expressing a dominant negative mutant of c-Jun N-terminal kinase 1 [JNK1(-)]. The inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase failed to prevent mercury-induced toxicity enhanced by SAAD. JNK1(-) cells were minimally susceptible to mercury in SAAD medium. These results demonstrated that SAAD potentiated cytotoxicity induced by arsenic or mercury and that activation of ERK1/2, p38 kinase and JNK1 was responsible for the potentiated arsenic toxicity, whereas the mercury toxicity enhanced by SAAD was mediated with the activity of JNK1.

  17. Protective function of pyridoxamine on retinal photoreceptor cells via activation of the p‑Erk1/2/Nrf2/Trx/ASK1 signalling pathway in diabetic mice.

    PubMed

    Ren, Xiang; Sun, Hong; Zhang, Chenghong; Li, Chen; Wang, Jinlei; Shen, Jie; Yu, Dong; Kong, Li

    2016-07-01

    The present study aimed to investigate the mechanisms that mediate the protective effects of pyridoxamine (PM) on light‑damaged retinal photoreceptor cells in diabetic mice. A high‑fat diet and streptozotocin were used to induce a mouse model of type II diabetes. During the experiment, mice were divided the mice into three types of group, as follows: Control groups (negative control and light‑damaged groups); experimental groups (diabetic and diabetic light‑damaged groups); and treatment groups (25, 50 and 100 mg/kg PM‑treated groups). Using hematoxylin‑eosin staining, the number of nuclear layer cells were counted. Western blotting and immunohistochemistry were performed to measure the levels of thioredoxin (Trx), phospho‑extracellular signal‑regulated kinase 1/2 (p‑Erk1/2), nuclear factor erythroid 2‑related factor 2 (Nrf2) and apoptosis signal‑regulating kinase 1 (ASK1). The photoreceptor cell count in the outer nuclear layer of the light‑damaged, diabetic control and diabetic light‑damaged groups were significantly reduced compared with the negative control group (P<0.001). The cell counts in the PM‑treated groups were significantly increased compared with the diabetic group (P<0.001). Compared with the negative control group, the light‑damaged, diabetic and diabetic light‑damaged groups exhibited significantly decreased Trx, p‑Erk1/2 and Nrf2 expression levels (P<0.001), and significantly increased ASK1 expression levels (P<0.001). However, in the PM‑treated groups, Trx, p‑Erk1/2 and Nrf2 expression levels were significantly increased (P<0.001), and ASK1 expression was significantly decreased (P<0.001). The results of the present study demonstrate that PM protects retinal photoreceptor cells against light damage in diabetic mice, and that its mechanism may be associated with the upregulation of Trx, p‑Erk1/2 and Nrf2 expression, and the downregulation of ASK1 expression. PMID:27177199

  18. A Potential Role for Mitochondrial Produced Reactive Oxygen Species in Salicylic Acid-Mediated Plant Acquired Thermotolerance.

    PubMed

    Nie, Shengjun; Yue, Haiyun; Xing, Da

    2015-10-15

    To characterize the function of salicylic acid (SA) in acquired thermotolerance, the effects of heat shock (HS) on wild-type and sid2 (for SA induction deficient 2) was investigated. After HS treatment, the survival ratio of sid2 mutant was lower than that of wild-type. However, pretreatment with hydrogen peroxide (H2O2) rescued the sid2 heat sensitivity. HsfA2 is a key component of acquired thermotolerance in Arabidopsis. The expression of HsfA2 induced by SA was highest among those of heat-inducible Hsfs (HsfA2, HsfA7a, HsfA3, HsfB1, and HsfB2) in response to HS. Furthermore, the application of AsA, an H2O2 scavenger, significantly reduced the expression level of HsfA2 induced by SA. Although SA enhanced the survival of sid2 mutant, no significant effect on the hsfA2 mutant was observed, suggesting that HsfA2 is responsible for SA-induced acquired thermotolerance as a downstream factor. Further, real-time PCR analysis revealed that after HS treatment, SA also up-regulated mRNA transcription of HS protein (Hsp) genes through AtHsfA2. Time course experiments showed an increase in the fluorescence intensity of DCF in the mitochondria occurred earlier than in other regions of the protoplasts in response to SA. The cytochrome reductase activity analysis in isolated mitochondria demonstrated that SA-induced mitochondrial ROS possibly originated from complex III in the respiration chain. Collectively, our data suggest that SA functions and acts upstream of AtHsfA2 in acquired thermotolerance, which requires a pathway with H2O2 production involved and is dependent on increased expression of Hsp genes. PMID:26099269

  19. Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

    PubMed Central

    Abou-Samra, Abdul B.

    2012-01-01

    Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0–40 min) of LLCP-K1 cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression. PMID:22414806

  20. 16-hydroxy-cleroda-3,13-dien-16,15-olide induced glioma cell autophagy via ROS generation and activation of p38 MAPK and ERK-1/2.

    PubMed

    Thiyagarajan, Varadharajan; Sivalingam, Kalai Selvi; Viswanadha, Vijaya Padma; Weng, Ching-Feng

    2016-07-01

    16-hydroxy-cleroda-3,13-dien-16,15-olide (HCD), a natural product isolated from medicinal plant Polyalthia longifolia exhibits anticancer activity through caspase-independent apoptosis in brain tumors, as previously reported. This study further attempted to investigate the involvement of HCD-induced autophagy in brain tumor cell lines neuroblastoma N18 and glioma C6 through the induction of reactive oxygen species (ROS) and the activation of p38 and ERK-1/2 pathway. The results demonstrated that HCD increased the hyper-generation of ROS and decreased cellular antioxidant enzymes, such as superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and glutathione s transferase (GST). Furthermore, HCD increased the expressions of autophagic marker proteins LC3-II and Beclin-1 in a time- and dose-dependent manner. Additionally, HCD was found to significantly induce p-p38 MAPK and p-ERK-1/2 proteins by Western blot, which implies that HCD is a potential therapeutic anticancer agent that exerts its activity through inducing ROS-mediation for the autophagy of brain tumor cells.

  1. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    PubMed Central

    Zhou, Yuanfei; Ren, Jiao; Song, Tongxing; Peng, Jian; Wei, Hongkui

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), methionine (Met), glutamine (Gln), and glycine (Gly), Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism. PMID:27727170

  2. ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells.

    PubMed

    Izawa, Yuki; Yoshizumi, Masanori; Fujita, Yoshiko; Ali, Nermin; Kanematsu, Yasuhisa; Ishizawa, Keisuke; Tsuchiya, Koichiro; Obata, Toshiyuki; Ebina, Yousuke; Tomita, Shuhei; Tamaki, Toshiaki

    2005-08-15

    Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser307 and Ser616. Ang II-induced Ser307 and Ser616 phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.

  3. TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation.

    PubMed

    Funcke, Jan-Bernd; Zoller, Verena; El Hay, Muad Abd; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2015-07-01

    Upon obesity, adipose tissue is excessively expanded and characterized by pathologic processes like hypoxia, fibrosis, and inflammation. Death ligands belonging to the TNF superfamily such as TNF-α are important contributors to these derangements and exert a pronounced influence on the metabolic and cellular homeostasis of adipose tissue. Here, we sought to identify the effect of the death ligand TNF-related apoptosis-inducing ligand (TRAIL) on the adipose tissue precursor cell pool and therefore investigated its influence on preadipocyte proliferation. Treatment of human preadipocytes with TRAIL resulted in a time- and dose-dependent increase in proliferation (EC50 3.4 ng/ml) comparable to IGF-1. Although no apoptosis was observed, TRAIL triggered a rapid cleavage of caspase-8 and -3. Neither inhibition of caspase activity by zVAD.fmk (20 µM) nor ablation of caspase-8 expression by lentivirus-delivered small hairpin RNA (shRNA) abolished the proliferative response. TRAIL triggered a delayed and sustained activation of ERK1/2, leaving Akt, p38, JNK, and NF-κB unaffected. Importantly, inhibition of ERK1/2 activation by PD0325901 (300 nM) or AZD6244 (5 or 10 µM) completely abolished the proliferative response. We thus reveal a hitherto unknown function of TRAIL in regulating adipose tissue homeostasis by promoting the proliferation of tissue-resident precursor cells. PMID:25857555

  4. Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation.

    PubMed

    Khan, Naim A; Nishimura, Kazuhiro; Aires, Virginie; Yamashita, Tomoko; Oaxaca-Castillo, David; Kashiwagi, Keiko; Igarashi, Kazuei

    2006-10-01

    Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

  5. Signaling through ERK1/2 controls myelin thickness during myelin repair in the adult central nervous system.

    PubMed

    Fyffe-Maricich, Sharyl L; Schott, Alexandra; Karl, Molly; Krasno, Janet; Miller, Robert H

    2013-11-20

    Oligodendrocytes, the myelin-forming cells of the CNS, exquisitely tailor the thickness of individual myelin sheaths to the diameter of their target axons to maximize the speed of action potential propagation, thus ensuring proper neuronal connectivity and function. Following demyelinating injuries to the adult CNS, newly formed oligodendrocytes frequently generate new myelin sheaths. Following episodes of demyelination such as those that occur in patients with multiple sclerosis, however, the matching of myelin thickness to axon diameter fails leaving remyelinated axons with thin myelin sheaths potentially compromising function and leaving axons vulnerable to damage. How oligodendrocytes determine the appropriate thickness of myelin for an axon of defined size during repair is unknown and identifying the signals that regulate myelin thickness has obvious therapeutic implications. Here, we show that sustained activation of extracellular-regulated kinases 1 and 2 (ERK1/2) in oligodendrocyte lineage cells results in accelerated myelin repair after injury, and is sufficient for the generation of thick myelin sheaths around remyelinated axons in the adult mouse spinal cord. Our findings suggest a model where ERK1/2 MAP kinase signaling acts as a myelin thickness rheostat that instructs oligodendrocytes to generate axon-appropriate quantities of myelin.

  6. TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation.

    PubMed

    Funcke, Jan-Bernd; Zoller, Verena; El Hay, Muad Abd; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2015-07-01

    Upon obesity, adipose tissue is excessively expanded and characterized by pathologic processes like hypoxia, fibrosis, and inflammation. Death ligands belonging to the TNF superfamily such as TNF-α are important contributors to these derangements and exert a pronounced influence on the metabolic and cellular homeostasis of adipose tissue. Here, we sought to identify the effect of the death ligand TNF-related apoptosis-inducing ligand (TRAIL) on the adipose tissue precursor cell pool and therefore investigated its influence on preadipocyte proliferation. Treatment of human preadipocytes with TRAIL resulted in a time- and dose-dependent increase in proliferation (EC50 3.4 ng/ml) comparable to IGF-1. Although no apoptosis was observed, TRAIL triggered a rapid cleavage of caspase-8 and -3. Neither inhibition of caspase activity by zVAD.fmk (20 µM) nor ablation of caspase-8 expression by lentivirus-delivered small hairpin RNA (shRNA) abolished the proliferative response. TRAIL triggered a delayed and sustained activation of ERK1/2, leaving Akt, p38, JNK, and NF-κB unaffected. Importantly, inhibition of ERK1/2 activation by PD0325901 (300 nM) or AZD6244 (5 or 10 µM) completely abolished the proliferative response. We thus reveal a hitherto unknown function of TRAIL in regulating adipose tissue homeostasis by promoting the proliferation of tissue-resident precursor cells.

  7. Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2-dependent mechanism.

    PubMed

    Hamdi, M M; Mutungi, G

    2011-07-15

    Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and α-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression.

  8. Intrathecally Transplanting Mesenchymal Stem Cells (MSCs) Activates ERK1/2 in Spinal Cords of Ischemia-Reperfusion Injury Rats and Improves Nerve Function

    PubMed Central

    Wang, Yonghong; Liu, He; Ma, Hong

    2016-01-01

    Background We investigated whether an intrathecal transplantation of mesenchymal stem cells (MSCs) activates extracellular adjusting protein kinase1 and 2(ERK1/2) in the spinal cords of rats following an ischemia-reperfusion injury, resulting in improved spinal cord function and inhibition of apoptosis. Material/Methods We observed the relationship between the activation of ERK1/2 in the rat spinal cord and intrathecal transplantation of MSCs, as well as the effect of U0126, a MEK1/2 (upstream protein of ERK1/2) inhibitor, on a spinal cord ischemia-reperfusion injury model in rats using Basso Beattie Bresnahan (BBB) scoring, somatosensory evoked potentials (SSEPs), immunohistochemistry, and Western blot analysis. Results After transplantation of MSCs, the lower limb motor function score increased, and the incubation period of SSEPs and amplitude were improved. Moreover, following transplantation of MSCs, Bcl2 expression increased, whereas Bax expression decreased after reperfusion. Transplantation of MSCs significantly enhanced pERK1/2 expression in the spinal cord, as well as pERK1/2 in immunoreactive cells located in the grey matter of the L4/5 levels of the spinal cord, following ischemia reperfusion injury in rats. The effective dose of U0126 required to inhibit pERK1/2 expression was 200 μg/kg. Bcl-2 decreased and the level of Bax expression increased in the spinal cord after ischemia reperfusion injury, and the protective effects of MSCs were attenuated. Conclusions Our findings suggest that intrathecal transplantation of MSCs activates ERK1/2 in the spinal cord following ischemia reperfusion injury, partially improves spinal cord function, and inhibits apoptosis in rats. PMID:27135658

  9. Intrathecally Transplanting Mesenchymal Stem Cells (MSCs) Activates ERK1/2 in Spinal Cords of Ischemia-Reperfusion Injury Rats and Improves Nerve Function.

    PubMed

    Wang, Yonghong; Liu, He; Ma, Hong

    2016-01-01

    BACKGROUND We investigated whether an intrathecal transplantation of mesenchymal stem cells (MSCs) activates extracellular adjusting protein kinase1 and 2(ERK1/2) in the spinal cords of rats following an ischemia-reperfusion injury, resulting in improved spinal cord function and inhibition of apoptosis. MATERIAL AND METHODS We observed the relationship between the activation of ERK1/2 in the rat spinal cord and intrathecal transplantation of MSCs, as well as the effect of U0126, a MEK1/2 (upstream protein of ERK1/2) inhibitor, on a spinal cord ischemia-reperfusion injury model in rats using Basso Beattie Bresnahan (BBB) scoring, somatosensory evoked potentials (SSEPs), immunohistochemistry, and Western blot analysis. RESULTS After transplantation of MSCs, the lower limb motor function score increased, and the incubation period of SSEPs and amplitude were improved. Moreover, following transplantation of MSCs, Bcl2 expression increased, whereas Bax expression decreased after reperfusion. Transplantation of MSCs significantly enhanced pERK1/2 expression in the spinal cord, as well as pERK1/2 in immunoreactive cells located in the grey matter of the L4/5 levels of the spinal cord, following ischemia reperfusion injury in rats. The effective dose of U0126 required to inhibit pERK1/2 expression was 200 µg/kg. Bcl-2 decreased and the level of Bax expression increased in the spinal cord after ischemia reperfusion injury, and the protective effects of MSCs were attenuated. CONCLUSIONS Our findings suggest that intrathecal transplantation of MSCs activates ERK1/2 in the spinal cord following ischemia reperfusion injury, partially improves spinal cord function, and inhibits apoptosis in rats. PMID:27135658

  10. Folic Acid Attenuates Vascular Endothelial Cell Injury Caused by Hypoxia via the Inhibition of ERK1/2/NOX4/ROS Pathway.

    PubMed

    Cheng, Fei; Lan, Jun; Xia, Wenhao; Tu, Chang; Chen, Benfa; Li, Shicheng; Pan, Weibiao

    2016-06-01

    Coronary artery disease is a disease with high morbidity and mortality, in which vascular endothelial dysfunction plays an important role. Hypoxia leads to the inflammation and oxidative stress in endothelial cells, which results in the endothelial injury. The present study was designed to investigate the protective effect and mechanism of folic acid on hypoxia-induced injury in human umbilical vein endothelial cells (HUVEC). Cell counting Kit was used to detect cell survival rate, and apoptotic cells were detected by Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured using dichloro-dihydro-fluorescein diacetate staining. Western blot was used to determine the protein expressions of extracellular signal protein kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2), NOX4 subunit of NAPDH and endothelial nitric oxide synthase (eNOS). Folic acid significantly increased the cell survival rate and decreased the apoptosis of HUVECs treated with folic acid compared with hypoxia-treated HUVEC. Folic acid also decreased ROS level, while it increased the nitrite content in HUVECs. In addition, folic acid decreased protein expressions of NOX4 and p-ERK1/2, while it increased the protein expression of eNOS in HUVECs. Furthermore, N-acetyl cysteine (NAC), the antioxidant, had similar effect on the cell survival rate and the apoptosis. In addition, DPI (NOX4 inhibitor) and U0126 (ERK1/2 inhibitor) rather than NAC decreased the protein expression of NOX4. NAC, DPI, and U0126 increased the protein expression of eNOS. Furthermore, U0126 rather than DPI and NAC decreased the protein expression of p-ERK1/2. Taken together, the results suggested that hypoxia decreased the cell survival rate and induced apoptosis via ERK1/2/NOX4/ROS pathway, which could be the target of folic acid in protecting the HUVECs from injury caused by hypoxia.

  11. Raloxifene analogue LY117018 suppresses oxidative stress-induced endothelial cell apoptosis through activation of ERK1/2 signaling pathway.

    PubMed

    Yu, Jing; Eto, Masato; Kozaki, Koichi; Akishita, Masahiro; Okabe, Tetsuro; Ouchi, Yasuyoshi

    2008-07-28

    A selective estrogen receptor modulator, raloxifene, has been shown to reduce cardiovascular events in relatively high-risk postmenopausal women with osteoporosis. However, the mechanisms by which raloxifene exerts a pharmacological effect on cardiovascular organs have not been fully elucidated. The present study was designed to examine whether the raloxifene analogue, 6-hydroxy-2-(p-hydroxyphenyl)-benzo(b) thien-3-yl-p-(2-(pyrrolidinyl)ethoxy phenyl ketone (LY117018), could inhibit apoptosis and to clarify the signaling pathway in vascular endothelial cells. LY117018 significantly inhibited hydrogen peroxide-induced apoptosis in bovine carotid artery endothelial cells. The anti-apoptotic effect of LY117018 was abolished by an estrogen receptor antagonist, 7alpha,7beta-(9[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl) estra-1,3,5(10)-triene-3,17-diol (ICI 182,780). Mitogen-activated protein kinases (MAPK), including p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase1/2 (ERK1/2), and Akt, have been shown to act as apoptotic or anti-apoptotic signals. Phosphorylation of p38, JNK, ERK1/2 and Akt was examined. LY117018 increased ERK1/2 phosphorylation but did not enhance the phosphorylation of p38, JNK, or Akt. The anti-apoptotic effect of LY117018 was prevented by treatment with 2-[2'-amino-3'-methoxyphenyl]-oxanaphthalen-4-one (PD98059), an upstream inhibitor of ERK1/2. LY117018 stimulated an increase in ERK1/2 phosphorylation, which was diminished by ICI 182,780. The activation of ERK/1/2 by LY117018 was not inhibited by the transcription inhibitor, actinomycin D. These results suggest that estrogen receptors and the ERK1/2 signaling pathway are involved in the anti-apoptotic action of LY117018 in vascular endothelial cells. PMID:18541231

  12. ERK1/2 and CaMKII-mediated events in memory formation: is 5HT regulation involved?

    PubMed

    Cammarota, Martín; Bevilaqua, Lia R; Medina, Jorge H; Izquierdo, Iván

    2008-12-16

    Activity-dependent changes in neuronal efficacy underlie the formation and storage of new memories. Several studies indicate that modification of the phosphorylation/activation state of different protein kinases localized in the synapses or the nucleus plays a critical role in the induction and maintenance of plastic mechanisms and in the consolidation of long-lasting memories. Here we review some of the more recent findings concerning the regulation of two of the main protein kinase groups involved in memory processes and in neuronal plasticity: Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the mitogen-activated protein kinase (MAPK) family. Since this issue of the journal is dedicated to serotonin (5HT) regulation of behavior, we will comment on the so far scanty, but significant, evidence for a role of 5HT in the regulation of CaMKII and MAPK.

  13. Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

    SciTech Connect

    D'Ambrosio, Steven M.; Han, Chunhua; Pan, Li; Douglas Kinghorn, A.; Ding, Haiming

    2011-06-10

    Highlights: {yields} The aliphatic acetogenins [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] (1) and [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate] (2) isolated from avocado fruit inhibit phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). {yields} Aliphatic acetogenin 2, but not 1, prevents EGF-induced activation of EGFR (Tyr1173). {yields} Combination of both aliphatic acetogenins synergistically inhibits c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation and human oral cancer cell proliferation. {yields} The potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins targeting two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. {yields} Providing a double hit on a critical cancer pathway such as EGFR/RAS/RAF/MEK/ERK1/2 by phytochemicals like those found in avocado fruit could lead to more effective approach toward cancer prevention. -- Abstract: Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not

  14. Rapamycin Reduced Ischemic Brain Damage in Diabetic Animals Is Associated with Suppressions of mTOR and ERK1/2 Signaling

    PubMed Central

    Liu, Ping; Yang, Xiao; Hei, Changchun; Meli, Yvonne; Niu, Jianguo; Sun, Tao; Li, P. Andy

    2016-01-01

    The objectives of the present study are to investigate the activation of mTOR and ERK1/2 signaling after cerebral ischemia in diabetic rats and to examine the neuroprotective effects of rapamycin. Ten minutes transient global cerebral ischemia was induced in straptozotocin-induced diabetic hyperglycemic rats and non-diabetic, euglycemic rats. Brain samples were harvested after 16 h of reperfusion. Rapamycin or vehicle was injected 1 month prior to the induction of ischemia. The results showed that diabetes increased ischemic neuronal cell death and associated with elevations of p-P70S6K and Ras/ERK1/2 and suppression of p-AMPKα. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and suppressed phosphorylation of P70S6K and ERK1/2. It is concluded that diabetes activates mTOR and ERK1/2 signaling pathways in rats subjected to transient cerebral ischemia and inhibition of mTOR by rapamycin reduces ischemic brain damage and suppresses the mTOR and ERK1/2 signaling in diabetic settings. PMID:27489506

  15. Rapamycin Reduced Ischemic Brain Damage in Diabetic Animals Is Associated with Suppressions of mTOR and ERK1/2 Signaling.

    PubMed

    Liu, Ping; Yang, Xiao; Hei, Changchun; Meli, Yvonne; Niu, Jianguo; Sun, Tao; Li, P Andy

    2016-01-01

    The objectives of the present study are to investigate the activation of mTOR and ERK1/2 signaling after cerebral ischemia in diabetic rats and to examine the neuroprotective effects of rapamycin. Ten minutes transient global cerebral ischemia was induced in straptozotocin-induced diabetic hyperglycemic rats and non-diabetic, euglycemic rats. Brain samples were harvested after 16 h of reperfusion. Rapamycin or vehicle was injected 1 month prior to the induction of ischemia. The results showed that diabetes increased ischemic neuronal cell death and associated with elevations of p-P70S6K and Ras/ERK1/2 and suppression of p-AMPKα. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and suppressed phosphorylation of P70S6K and ERK1/2. It is concluded that diabetes activates mTOR and ERK1/2 signaling pathways in rats subjected to transient cerebral ischemia and inhibition of mTOR by rapamycin reduces ischemic brain damage and suppresses the mTOR and ERK1/2 signaling in diabetic settings. PMID:27489506

  16. Silica nanoparticles increase human adipose tissue-derived stem cell proliferation through ERK1/2 activation

    PubMed Central

    Kim, Ki Joo; Joe, Young Ae; Kim, Min Kyoung; Lee, Su Jin; Ryu, Yeon Hee; Cho, Dong-Woo; Rhie, Jong Won

    2015-01-01

    Background Silicon dioxide composites have been found to enhance the mechanical properties of scaffolds and to support growth of human adipose tissue-derived stem cells (hADSCs) both in vitro and in vivo. Silica (silicon dioxide alone) exists as differently sized particles when suspended in culture medium, but it is not clear whether particle size influences the beneficial effect of silicon dioxide on hADSCs. In this study, we examined the effect of different sized particles on growth and mitogen-activated protein kinase signaling in hADSCs. Methods Silica gel was prepared by a chemical reaction using hydrochloric acid and sodium silicate, washed, sterilized, and suspended in serum-free culture medium for 48 hours, and then sequentially filtered through a 0.22 μm filter (filtrate containing nanoparticles smaller than 220 nm; silica NPs). hADSCs were incubated with silica NPs or 3 μm silica microparticles (MPs), examined by transmission electron microscopy, and assayed for cell proliferation, apoptosis, and mitogen-activated protein kinase signaling. Results Eighty-nine percent of the silica NPs were around 50–120 nm in size. When hADSCs were treated with the study particles, silica NPs were observed in endocytosed vacuoles in the cytosol of hADSCs, but silica MPs showed no cell entry. Silica NPs increased the proliferation of hADSCs, but silica MPs had no significant effect in this regard. Instead, silica MPs induced slight apoptosis. Silica NPs increased phosphorylation of extracellular signal-related kinase (ERK)1/2, while silica MPs increased phosphorylation of p38. Silica NPs had no effect on phosphorylation of Janus kinase or p38. Pretreatment with PD98059, a MEK inhibitor, prevented the ERK1/2 phosphorylation and proliferation induced by silica NPs. Conclusion Scaffolds containing silicon dioxide for tissue engineering may enhance cell growth through ERK1/2 activation only when NPs around 50–120 nm in size are included, and single component silica

  17. In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.

    PubMed

    Lebraud, Honorine; Wright, David J; East, Charlotte E; Holding, Finn P; O'Reilly, Marc; Heightman, Tom D

    2016-08-16

    In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition. PMID:27385078

  18. TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

    SciTech Connect

    Sun, Jing; Liu, Su-zhi; Lin, Yan; Cao, Xiao-pan; Liu, Jia-ming

    2014-01-17

    Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significant when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy.

  19. S-Adenosylmethionine Affects ERK1/2 and Stat3 Pathways and Induces Apotosis in Osteosarcoma Cells.

    PubMed

    Ilisso, Concetta Paola; Sapio, Luigi; Delle Cave, Donatella; Illiano, Michela; Spina, Annamaria; Cacciapuoti, Giovanna; Naviglio, Silvio; Porcelli, Marina

    2016-02-01

    Osteosarcoma is a very aggressive bone tumor. Its clinical outcome remains discouraging despite intensive surgery, radiotherapy, and chemotherapy. Thus, novel therapeutic approaches are demanded. S-Adenosylmethionine (AdoMet) is a naturally occurring molecule that is synthesized in our body by methionine adenosyltransferase isoenzymes and is also available as a nutritional supplement. AdoMet is the principal methyl donor in numerous methylation reactions and is involved in many biological functions. Interestingly, AdoMet has been shown to exert antiproliferative action in various cancer cells. However, the underlying molecular mechanisms are just starting to be studied. Here, we investigated the effects of AdoMet on the proliferation of osteosarcoma U2OS cells and the underlying mechanisms. We carried out direct cell number counting, MTT and flow cytometry-based assays, and immunoblotting experiments in response to AdoMet treatment. We found that AdoMet strongly inhibits proliferation of U2OS cells by slowing-down cell cycle progression and by inducing apoptosis. We also report that AdoMet consistently causes an increase of p53 and p21 cell-cycle inhibitor, a decrease of cyclin A and cyclin E protein levels, and a marked increase of pro-apoptotic Bax/Bcl-2 ratio, with caspase-3 activation and PARP cleavage. Moreover, the AdoMet-induced antiproliferative effects were dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels. Altogether, our data enforce the evidence of AdoMet acting as a biomolecule with antiproliferative action in osteosarcoma cells, capable of down-regulating ERK1/2 and STAT3 pathways leading to cell cycle inhibition and apoptosis, and provide a rationale for the possible use of AdoMet in osteosarcoma therapy.

  20. Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells

    PubMed Central

    Chuang, Dennis Y.; Cui, Jiankun; Simonyi, Agnes; Engel, Victoria A.; Chen, Shanyan; Fritsche, Kevin L.; Thomas, Andrew L.; Applequist, Wendy L.; Folk, William R.; Lubahn, Dennis B.; Sun, Albert Y.; Sun, Grace Y.

    2014-01-01

    Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R. PMID:25324465

  1. Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway.

    PubMed

    Wang, Qin; Chuikov, Sergei; Taitano, Sophina; Wu, Qi; Rastogi, Arjun; Tuck, Samuel J; Corey, Joseph M; Lundy, Steven K; Mao-Draayer, Yang

    2015-01-01

    Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS). PMID:26090715

  2. Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway.

    PubMed

    Wang, Qin; Chuikov, Sergei; Taitano, Sophina; Wu, Qi; Rastogi, Arjun; Tuck, Samuel J; Corey, Joseph M; Lundy, Steven K; Mao-Draayer, Yang

    2015-01-01

    Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS).

  3. Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation

    PubMed Central

    Rosenberger, Simone; Arce, Johanna De-Castro; Langbein, Lutz; Steenbergen, Renske D. M.; Rösl, Frank

    2010-01-01

    Certain types of human papillomaviruses (HPVs) are etiologically linked to cervical cancer. Their transforming capacity is encoded by a polycistronic premRNA, where alternative splicing leads to the translation of functional distinct proteins such as E6, E6*, and E7. Here we show that splicing of HPV16 E6/E7 ORF cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, whereas depletion of EGF, or treatment with EGF receptor (EGFR) neutralizing antibodies or the EGFR inhibitor tyrphostin AG1478, resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability, or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Because there is a natural gradient of EGF and EGF receptor expression in the stratified epithelium, it is reasonable to assume that EGF modulates E6/E7 splicing during the viral life cycle and transformation. PMID:20351270

  4. Signaling from the Human Melanocortin 1 Receptor to ERK1 and ERK2 Mitogen-Activated Protein Kinases Involves Transactivation of cKIT

    PubMed Central

    Herraiz, Cecilia; Journé, Fabrice; Abdel-Malek, Zalfa; Ghanem, Ghanem; Jiménez-Cervantes, Celia; García-Borrón, José C.

    2011-01-01

    Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. Upon stimulation by αMSH, MC1R triggers the cAMP and ERK1/ERK2 MAPK pathways. In mouse melanocytes, ERK activation by αMSH binding to Mc1r depends on cAMP, and melanocytes are considered a paradigm for cAMP-dependent ERK activation. However, human MC1R variants associated with red hair, fair skin [red hair color (RHC) phenotype], and increased skin cancer risk display reduced cAMP signaling but activate ERKs as efficiently as wild type in heterologous cells, suggesting independent signaling to ERKs and cAMP in human melanocytes. We show that MC1R signaling activated the ERK pathway in normal human melanocytes and melanoma cells expressing physiological levels of endogenous RHC variants. ERK activation was comparable for wild-type and mutant MC1R and was independent on cAMP because it was neither triggered by stimulation of cAMP synthesis with forskolin nor blocked by the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine. Stimulation of MC1R with αMSH did not lead to protein kinase C activation and ERK activation was unaffected by protein kinase C inhibitors. Conversely, pharmacological interference, small interfering RNA studies, expression profiles, and functional reconstitution experiments showed that αMSH-induced ERK activation resulted from Src tyrosine kinase-mediated transactivation of the stem cell factor receptor, a receptor tyrosine kinase essential for proliferation, differentiation, and survival of melanocyte precursors, thus demonstrating a functional link between the stem cell factor receptor and MC1R. Moreover, this transactivation phenomenon is unique because it is unaffected by natural mutations impairing canonical MC1R signaling through the cAMP pathway. PMID:21084381

  5. Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

    PubMed Central

    Jazirehi, Ali R.; Economou, James S.

    2012-01-01

    Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

  6. Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases.

    PubMed

    Violet, Pierre-Christian; Billon-Denis, Emmanuelle; Robin, Philippe

    2012-11-01

    The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.

  7. Abrogated thioredoxin system causes increased sensitivity to TNF-α-induced apoptosis via enrichment of p-ERK 1/2 in the nucleus.

    PubMed

    Yoo, Min-Hyuk; Carlson, Bradley A; Gladyshev, Vadim N; Hatfield, Dolph L

    2013-01-01

    Thioredoxin (Trx) and thioredoxin reductase 1 (TR1) are among the major redox regulators in mammalian cells and have a wide variety of roles, including removal of intracellular reactive oxygen species (ROS) and prevention of cell death. Tumor necrosis factor-α (TNF-α) induces cancer cell death. Although ROS have been proposed to participate in this process, the role of the thioredoxin system in TNF-α stimulated cell death remains unclear. We investigated the possibility that the thioredoxin system protects against TNF-α-induced cancer cell death by examining whether TR1/Trx1 status controls TNF-α-induced apoptosis in EMT6 murine breast cancer cells. TR1-deficient cells were more sensitive to TNF-α than control cells. Increased sensitivity to TNF-α was most pronounced in Trx1-deficient cells. TNF-α-induced nuclear localization of phosphorylated ERK 1/2 (p-ERK 1/2) correlated with increased apoptosis in TR1- and Trx1-deficient cells, suggesting a pro-apoptotic role for nuclear p-ERK 1/2 in TNF-α-induced apoptosis. In addition, phosphoinositide 3-kinase (PI3K) inhibition dramatically reduced TNF-α-stimulated apoptosis and nuclear localization of p-ERK 1/2. In contrast, inhibition of ROS, MEK, JNK, or p38 did not significantly alter p-ERK 1/2 localization or apoptosis in TR1- and Trx1-deficient cells compared to control cells. Further, NF-κB p65 localization was not changed in TR1- and Trx1-deficient cells in response to TNF-α relative to control cells. Our data suggest that the thioredoxin system plays a critical role in protecting against TNF-α-induced apoptosis by regulating the levels of nuclear p-ERK 1/2 in a PI3K-dependent manner.

  8. Involvement of MEK-ERK1-2 pathway in the anti-oxidant response in C6 glioma cells after diesel exhaust particles exposure.

    PubMed

    Farina, Francesca; Milani, Chiara; Botto, Laura; Lonati, Elena; Bulbarelli, Alessandra; Palestini, Paola

    2016-05-27

    Ultrafine particles translocate to the central nervous system and activate oxidative stress-related pathways. The transcription factor Nrf2 activation by ERK1-2 has been suggested as a key regulator of cellular response to oxidative stress. C6 glioma cells have been treated with different doses of diesel exhaust particles (25μg/ml, DEP25, and 50μg/ml, DEP50), for different times. Cells have been screened for oxidative stress and inflammatory markers, and for the activation of the MEK-ERK1-2 pathway. The same markers have been examined after inhibition of MEK, the kinase upstream to ERK1-2. 3h and 24h of DEP25 and DEP50 induced a significant increase in HO-1 levels. After 24h, DEP25 and DEP50 induced an increase in HO-1 and Cyp1b1 levels, while increase in OGG1 level was observed only with DEP25. After 5h of treatment with DEP25, ERK1-2 resulted phosphorylated, concomitantly with a significant increase in HO-1 levels, no changes in iNOS levels, and decreased levels of anti-oxidant enzymes. After treatment with MEK inhibitor U0126, ERK1-2 showed no activation, with a consequent decrease in Nrf2, no increase in HO-1 and a significant increase of iNOS. MEK inhibitor is able to deplete anti-oxidant enzymes. In conclusion, the MEK-ERK1-2 pathway is involved in regulating the anti-oxidant strategies to compensate the oxidative status induced by DEP treatment. PMID:27091075

  9. Effects of Wenyangzhenshuai Granule on ERK1/2 and ERK5 activity in the myocardial tissue in a rabbit model of adriamycin-induced chronic heart failure

    PubMed Central

    Chen, Xinyu; Cai, Huzhi; Chen, Qingyang; Xie, Haibo; Liu, Yuemei; Lu, Qing; Tang, Yanping

    2015-01-01

    Objective: To elucidate the effects of Wenyangzhenshuai granule on expression of extracellular signal-regulated kinase 1/2 (ERK1/2) and 5 (ERK5) in the myocardial tissue using a rabbit model of adriamycin-induced chronic heart failure. Materials and methods: Rabbits were divided into heart failure positive control, adriamycin injection, and adriamycin injection with Wenyangzhenshuai treatment (low, medium and high dose) groups. Cardiac function and cardiac hypotrophy were measured in all groups. Besides, myocardial expression of ERK1/2 and ERK5 phosphorylation were evaluated by Western blotting and ERK1/2 and ERK5 mRNA levels by RT-PCR. The cardiac structure and cardiac function were also compared using histology staining and electron microscope. Results: Adriamycin injection led to cardiac failure reflected by decreased left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), E/A ratio, and increased cardiac hypertrophy, both of which have been improved by Wenyangzhenshuai granule treatment (all P<0.05). Mechanistically, increased P-ERK1/2 and decreased P-ERK5 levels were observed in myocardial tissues of mice treated with Adriamycin for 8 weeks. However, such signaling change could be partially corrected by Wenyangzhenshuai treatment. In addition, no significant difference was detected in the expression of ERK1/2 and ERK5 mRNA levels between adriamycin injection groups and Wenyangzhenshuai treatment groups (P>0.05), indicating an alteration in the activity/phosphorylation levels of these proteins instead of the transcription levels. Conclusion: we found a beneficial effect of Wenyangzhenshuai treatment in partially decelerating the progression of CHF. Such effect was probably through the role of Wenyangzhenchuan in diminishing p-ERK1/2 and raising p-ERK5 level in myocardial tissue. PMID:26884996

  10. Resilience to audiogenic seizures is associated with p-ERK1/2 dephosphorylation in the subiculum of Fmr1 knockout mice

    PubMed Central

    Curia, Giulia; Gualtieri, Fabio; Bartolomeo, Regina; Vezzali, Riccardo; Biagini, Giuseppe

    2013-01-01

    Young, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P < 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P < 0.05 vs. WT) and CA3 (P < 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P < 0.05 vs. WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (–75%, P < 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1

  11. GIT1Y321 phosphorylation is required for ERK1/2- and PDGF-dependent VEGF secretion from osteoblasts to promote angiogenesis and bone healing.

    PubMed

    Rui, Ze; Li, Xiang; Fan, Jin; Ren, Yongxin; Yuan, Yufeng; Hua, Zhengzhe; Zhang, Ning; Yin, Guoyong

    2012-10-01

    Bone healing depends on vascular endothelial growth factor (VEGF) secretion from osteoblasts to promote angiogenesis. We examined the influence of the tyrosine 321 site of G protein-coupled receptor kinase interacting protein 1 (GIT1) on platelet-derived growth factor (PDGF)-induced VEGF synthesis in vitro and on bone healing in vivo. Cultured osteoblasts were prepared from calvaria of 1-2-day-old rats. The phospho-activation of extracellular signal-regulated kinases 1/2 (ERK1/2), GIT1, the interaction between GIT1 and ERK1/2, and VEGF mRNA expression were measured in response to PDGF. In addition, PDGF was applied following pretreatment with the MEK1/2 inhibitor PD98059 or the Src inhibitor PP2. We mutated tyrosines 293 or 321 of GIT1 individually to phenylalanine (mutants GIT1Y293F and GIT1Y321F) and incorporated these mutants and native GIT1 into lentivirus vectors. The relationship between GIT1 and ERK1/2, and VEGF mRNA expression in cultured osteoblasts were detected after infection with GIT1WT-, GIT1Y293F- and GIT1Y321F-expressing lentivirus in response to PDGF. Bone healing and expression of VEGF and the angiogenic marker PECAM-1 were evaluated after infection at the fracture site. Activation of ERK1/2 by phosphorylation, GIT1 tyrosine phosphorylation, GIT1-ERK1/2 interaction, and VEGF mRNA expression were all significantly increased in osteoblasts after PDGF stimulation, but all responses were dramatically inhibited by pretreatment with PD98059. Tyrosine phosphorylation, GIT1 interaction with ERK1/2, and VEGF mRNA expression were dramatically inhibited by pretreatment with PP2 or infection with GIT1Y321F-expressing lentivirus. Expression of VEGF and PECAM-1 was significantly lower at the fracture sites infected with GIT1Y321F-expressing lentivirus and bone healing was significantly delayed compared to fracture sites infected with GIT1WT. In conclusion, tyrosine 321 of GIT1 is a critical phosphorylation site for GIT1 interaction with ERK1/2, regulation of

  12. The fruit juice of Morinda citrifolia (noni) downregulates HIF-1α protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.

    PubMed

    Jang, Byeong-Churl

    2012-03-01

    High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1α, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1α protein but did not affect HIF-1α mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1α upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2α (eIF-2α) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1α protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1α protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1α protein by desferoxamine or interleukin-1β (IL-1β), another HIF-1α inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1α protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1α overexpression play pathogenic roles.

  13. Key role of the ERK1/2 MAPK pathway in the transcriptional regulation of the Stearoyl-CoA Desaturase (SCD1) gene expression in response to leptin.

    PubMed

    Mauvoisin, Daniel; Prévost, Michèle; Ducheix, Simon; Arnaud, Marie-Pierre; Mounier, Catherine

    2010-05-01

    Stearoyl-CoA Desaturase-1 (SCD1) is the rate limiting enzyme catalyzing the synthesis of monounsaturated fatty acids. Variation of SCD1 activity and the ratio of saturated to unsaturated fatty acids have been implicated in a variety of diseases including obesity, type II diabetes and cancers. In liver, many factors regulate SCD1 expression including dietary and hormonal factors such as insulin and leptin. We previously showed in hepatic cells that insulin acts through the PI3K and mTOR pathways to upregulate SCD1 expression. In the present study, using HepG2 cells, we characterized the signaling pathway mediating the leptin inhibitory response on SCD1 gene expression. We showed that leptin inhibits SCD1 at the transcriptional level. Inhibition of the ERK1/2 MAPK pathway with the PD98059 reverses the effect of leptin on SCD1 expression. Our data also demonstrated that the effect of leptin is entirely independent of the effect of insulin. Using the pharmaceutical inhibitors Ag490 and SL0101, we showed that the inhibitory effect of leptin is also mediated by the Janus Kinase 2 (Jak2) and p90RSK. EMSA and transfection experiments suggest a key role for the Sp1 transcription factor, which in turn may compete for the binding of other transcription factors such as AP-1, leading to the inhibition of SCD1 transcription. Taken together, our observations showed that, independently of insulin action, leptin exerts an inhibitory effect on SCD1 transcription via a signaling pathway implicating Jak2, ERK1/2, and p90RSK which probably targets the downstream transcription factor Sp1 on the SCD1 promoter.

  14. Acquired Tumor Cell Radiation Resistance at the Treatment Site Is Mediated Through Radiation-Orchestrated Intercellular Communication

    SciTech Connect

    Aravindan, Natarajan; Aravindan, Sheeja; Pandian, Vijayabaskar; Khan, Faizan H.; Ramraj, Satish Kumar; Natt, Praveen; Natarajan, Mohan

    2014-03-01

    Purpose: Radiation resistance induced in cancer cells that survive after radiation therapy (RT) could be associated with increased radiation protection, limiting the therapeutic benefit of radiation. Herein we investigated the sequential mechanistic molecular orchestration involved in radiation-induced radiation protection in tumor cells. Results: Radiation, both in the low-dose irradiation (LDIR) range (10, 50, or 100 cGy) or at a higher, challenge dose IR (CDIR), 4 Gy, induced dose-dependent and sustained NFκB-DNA binding activity. However, a robust and consistent increase was seen in CDIR-induced NFκB activity, decreased DNA fragmentation, apoptosis, and cytotoxicity and attenuation of CDIR-inhibited clonal expansion when the cells were primed with LDIR prior to challenge dose. Furthermore, NFκB manipulation studies with small interfering RNA (siRNA) silencing or p50/p65 overexpression unveiled the influence of LDIR-activated NFκB in regulating CDIR-induced DNA fragmentation and apoptosis. LDIR significantly increased the transactivation/translation of the radiation-responsive factors tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), cMYC, and SOD2. Coculture experiments exhibit LDIR-influenced radiation protection and increases in cellular expression, secretion, and activation of radiation-responsive molecules in bystander cells. Individual gene-silencing approach with siRNAs coupled with coculture studies showed the influence of LDIR-modulated TNF-α, IL-1α, cMYC, and SOD2 in induced radiation protection in bystander cells. NFκB inhibition/overexpression studies coupled with coculture experiments demonstrated that TNF-α, IL-1α, cMYC, and SOD2 are selectively regulated by LDIR-induced NFκB. Conclusions: Together, these data strongly suggest that scattered LDIR-induced NFκB-dependent TNF-α, IL-1α, cMYC, and SOD2 mediate radiation protection to the subsequent challenge dose in tumor cells.

  15. Induction of Connective Tissue Growth Factor Expression by Hypoxia in Human Lung Fibroblasts via the MEKK1/MEK1/ERK1/GLI-1/GLI-2 and AP-1 Pathways

    PubMed Central

    Cheng, Yi; Lin, Chien-huang; Chen, Jing-Yun; Li, Chien-Hua; Liu, Yu-Tin; Chen, Bing-Chang

    2016-01-01

    GLI-2 siRNA. Overall, these data implied that the MEKK1/MEK1/ERK1/GLI-1/GLI-2, and AP-1 pathways mediated hypoxia-induced CTGF expression in human lung fibroblasts. Furthermore, GLI-1 and GLI-2 found to be involved in hypoxia-induced α-SMA and collagen expression. PMID:27486656

  16. Suppressor of cytokine signaling 1 inhibits IFN-gamma inflammatory signaling in human keratinocytes by sustaining ERK1/2 activation.

    PubMed

    Madonna, Stefania; Scarponi, Claudia; De Pità, Ornella; Albanesi, Cristina

    2008-09-01

    IFN-gamma is a pleiotropic cytokine importantly involved in the development of skin inflammatory responses. Epidermal keratinocytes are extremely susceptible to IFN-gamma action, but, once transduced with the suppressors of cytokine signaling (SOCS)1 molecule, they can no longer express a number of IFN-gamma-inducible signal transducer and activator of transcription (STAT)1-dependent genes. Extracellular-signal-regulated kinase (ERK)1/2 pathway is also involved in the protection of keratinocytes from the proinflammatory effect of IFN-gamma. Here we show that, after IFN-gamma stimulation, SOCS1 inhibited IFN-gamma receptor and STAT1 phosphorylation but maintained ERK1/2 activation. SOCS1 was also necessary for the IFN-gamma-induced RAS and Raf-1 activities in keratinocytes. The enhanced ERK1/2 pathway in SOCS1-overexpressing keratinocytes was in part responsible for their inability to respond to IFN-gamma, in terms of CXCL10 and CCL2 production, and for the high production of CXCL8. Moreover, SOCS1 interacted with the RAS inhibitor p120 RasGAP and promoted its degradation after IFN-gamma stimulation. We hypothesize that SOCS1 functions as suppressor of IFN-gamma signaling, not only by inhibiting STAT1 activation but also by sustaining ERK1/2-dependent antiinflammatory pathways.

  17. Modulation of human gingival fibroblast adhesion, morphology, tyrosine phosphorylation, and ERK 1/2 localization on polished, grooved and SLA substratum topographies.

    PubMed

    Kokubu, Eitoyo; Hamilton, Douglas W; Inoue, Takashi; Brunette, Donald M

    2009-12-01

    Attachment of connective tissue to dental implants, which is influenced by surface topography, is an important determinant of implant success. Approaches employed to alter topography include acid etching or blasting to produce roughened surfaces, and production of precisely defined topographies using microfabrication techniques. The aim of this study was to assess the influence of polished, microgrooved, and sand-blasted, large grit, acid-etched (SLA) topographies on fibroblast adhesion, morphology, activation, and ERK 1/2 phosphorylation and localization. Human gingival fibroblasts (HGFs) spread on all tested surfaces within 2 h, and topography influenced the pattern of phosphotyrosine localization. Fibrillar adhesion formation was prominent in HGFs cultured on microgrooves and SLA at 24 h compared with smooth. No significant difference in ERK 1/2 phosphorylation was observed at 2 or 24 h, but nuclear localization depended on culture time and substratum topography. Nuclear localization of ERK 1/2 occurred at 2 h on polished surfaces, but was not evident at 1 week. In contrast, cells on SLA and grooved surfaces did not exhibit nuclear localization of ERK 1/2 at early times, but did at 1 week. The results of this study suggest that rough and microfabricated topographies influence fibroblast adhesion and intracellular signaling through focal adhesion/integrin-dependent mechanisms in a time-dependent manner.

  18. GM1 ganglioside activates ERK1/2 and Akt downstream of Trk tyrosine kinase and protects PC12 cells against hydrogen peroxide toxicity.

    PubMed

    Zakharova, Irina O; Sokolova, Tatyana V; Vlasova, Yulia A; Furaev, Victor V; Rychkova, Maria P; Avrova, Natalia F

    2014-11-01

    Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na(+),K(+)-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.

  19. Modulation of Akt and ERK1/2 Pathways by Resveratrol in Chronic Myelogenous Leukemia (CML) Cells Results in the Downregulation of Hsp70

    PubMed Central

    Raha, Sanghamitra

    2010-01-01

    Background Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1. Methodology/Principal Findings Cells exposed to 40µM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3β. Active non-phosphorylated Gsk3β rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3β activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells. Conclusion/Significance Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings

  20. Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells

    SciTech Connect

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Glisic, Branka; Kaisarevic, Sonja; Kovacevic, Radmila; Andric, Nebojsa

    2013-07-01

    Worldwide used herbicide atrazine is linked to reproductive dysfunction in females. In this study, we investigated the effects and the mechanism of atrazine action in the ovary using a primary culture of immature granulosa cells. In granulosa cells, follicle-stimulating hormone (FSH) activates both cyclic adenosine monophosphate (cAMP) and extracellular-regulated kinase 1/2 (ERK1/2) cascades, with cAMP pathway being more important for luteinizing hormone receptor (LHR) and aromatase (CYP19A1) mRNA expression. We report that 48 h after atrazine exposure the FSH-stimulated LHR and CYP19A1 mRNA expression and estradiol synthesis were decreased, with LHR mRNA being more sensitive to atrazine than CYP19A1 mRNA. Inadequate acquisition of LHR in the FSH-stimulated and atrazine-exposed granulosa cells renders human chorionic gonadotropin (hCG) ineffective to stimulate amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr) mRNA expression, suggesting anti-ovulatory effect of atrazine. To dissect the signaling cascade involved in atrazine action in granulosa cells, we used U0126, a pharmacological inhibitor of ERK1/2. U0126 prevents atrazine-induced decrease in LHR and CYP19A1 mRNA levels and estradiol production in the FSH-stimulated granulosa cells. ERK1/2 inactivation restores the ability of hCG to induce expression of the ovulatory genes in atrazine-exposed granulosa cells. Cell-based ELISA assay revealed that atrazine does not change the FSH-stimulated ERK1/2 phosphorylation in granulosa cells. The results from this study reveal that atrazine does not affect but requires ERK1/2 phosphorylation to cause decrease in the FSH-induced LHR and CYP19A1 mRNA levels and estradiol production in immature granulosa cells, thus compromising ovulation and female fertility. - Highlights: • Atrazine inhibits estradiol production in FSH-stimulated granulosa cells. • Atrazine inhibits LHR and Cyp19a1 mRNA expression in FSH-stimulated granulosa cells. • Atrazine

  1. Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 Is associated with the downregulation of peroxisome proliferator-activated receptor (PPAR)-γ during polymicrobial sepsis.

    PubMed

    Kaplan, Jennifer M; Hake, Paul W; Denenberg, Alvin; Nowell, Marchele; Piraino, Giovanna; Zingarelli, Basilia

    2010-01-01

    Peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury.

  2. The hyaluronan receptors CD44 and RHAMM (CD168) form complexeswith ERK1,2, which sustain high basal motility in breast cancercells

    SciTech Connect

    Hamilton, Sara R.; Fard, Shireen F.; Paiwand, Frouz F.; Tolg,Cornelia; Veiseh, Mandana; Wang, Chao; McCarthy, James B.; Bissell, MinaJ.; Koropatnick, James; Turley, Eva A.

    2007-03-28

    CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a non-integral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture and its expression is strongly upregulated in diseases like arthritis and aggressive cancers. Here we describe an autocrine mechanism utilizing cell surface Rhamm/CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines. This mechanism requires endogenous hyaluronan synthesis and the formation of Rhamm/CD44/ERK1, 2 complexes. Motile/ invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit elevated basal activation of ERK1, 2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm and ERK1, 2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Rapid motility of the invasive cell lines requires interaction of hyaluronan with cells, activation of ERK1, 2 and the participation of both cell surface CD44 and Rhamm. Combinations of anti-CD44, anti-Rhamm antibodies and a MEK1 inhibitor (PD098059) have less-than-additive blocking effects, suggesting action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent, autocrine mechanism to coordinate sustained signaling through ERK1, 2 leading to high basal motility of invasive breast cancer cells. Since CD44/Rhamm complexes are not evident in less motile cells, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, in this case cell surface Rhamm.

  3. Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2.

    PubMed

    Gao, Gongming; Shen, Nan; Jiang, Xuefeng; Sun, Huiqing; Xu, Nanwei; Zhou, Dong; Nong, Luming; Ren, Kewei

    2016-01-15

    The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

  4. Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

    PubMed Central

    Tong, An-li; Wang, Fen; Cui, Yun-ying; Li, Chun-yan; Li, Yu-xiu

    2016-01-01

    The cross talk between angiotensin II (Ang II) and insulin has been described mainly in cardiovascular cells, hepatocytes, adipocytes, and so forth, and to date no such cross talk was reported in adrenal. In this study, we examined the interaction between Ang II and insulin/IGF-1 in ERK and AKT signaling pathways and expression of steroidogenic enzymes in H295R cells. Compared to the control, 100 nM Ang II increased phospho-ERK1/2 approximately 3-fold. Insulin (100 nM) or IGF-1 (10 nM) alone raised phospho-ERK1/2 1.8- and 1.5-fold, respectively, while, after pretreatment with 100 nM Ang II for 30 min, insulin (100 nM) or IGF-1 (10 nM) elevated phospho-ERK1/2 level 8- and 7-fold, respectively. The synergistic effect of Ang II and insulin/IGF-1 on ERK1/2 activation was inhibited by selective AT1 receptor blocker, PKC inhibitor, and MEK1/2 inhibitor. Ang II marginally suppressed AKT activation under the basal condition, while it had no effect on phospho-AKT induced by insulin/IGF-1. Ang II significantly stimulated mRNA expression of CYP11B1 and CYP11B2, and such stimulatory effects were enhanced when cells were cotreated with insulin/IGF-1. We are led to conclude that Ang II in combination with insulin/IGF-1 had an evident synergistic stimulatory effect on ERK1/2 activation in H295R cells and the effect may be responsible for the enhanced steroid hormone production induced by Ang II plus insulin/IGF-1. PMID:27293433

  5. A selective estrogen receptor modulator inhibits TNF-alpha-induced apoptosis by activating ERK1/2 signaling pathway in vascular endothelial cells.

    PubMed

    Yu, Jing; Eto, Masato; Akishita, Masahiro; Okabe, Tetsuro; Ouchi, Yasuyoshi

    2009-07-01

    Tumor necrosis factor (TNF-alpha) is a pleiotropic cytokine exerting both inflammatory and cell death activity and is thought to play a role in the pathogenesis of atherosclerosis. The present study was designed to examine whether the raloxifene analogue, LY117018 could inhibit TNF-alpha-induced apoptosis in vascular endothelial cells and to clarify the involved mechanisms. Apoptosis of endothelial cells was determined by DNA fragmentation assay and the activation of caspase-3. LY117018 significantly inhibited TNF-alpha-induced caspase-3 activation and cell DNA fragmentation levels in bovine carotid artery endothelial cells. The inhibitory effect of LY117018 was abolished by an estrogen receptor antagonist ICI 182,780. p38 MAPK, JNK, ERK1/2 and Akt have been shown to act as apoptotic or anti-apoptotic signals. TNF-alpha stimulated the phosphorylation levels of p38 MAPK, JNK, ERK1/2 and Akt in vascular endothelial cells. TNF-alpha-induced apoptosis was significantly decreased by SB203580, a p38 MAPK inhibitor or SP600125, a JNK inhibitor, but was enhanced by an ERK1/2 pathway inhibitor, PD98059 or a PI3-kinase/Akt pathway inhibitor, wortmannin. The anti-apoptotic effect of LY117018 was abrogated only by PD98059 but was not affected by the inhibitors for p38 MAPK, JNK, or Akt. LY117018 stimulated the further increase in phosphorylation of ERK1/2 in TNF-alpha treated endothelial cells but it did not affect phosphorylation levels of p38 MAPK, JNK or Akt. These results suggest that LY 110718 prevents caspase-3 dependent apoptosis induced by TNF-alpha in vascular endothelial cells through activation of the estrogen receptors and the ERK1/2 signaling pathway. PMID:19275968

  6. Cadmium at nanomolar concentrations activates Raf-MEK-ERK1/2 MAPKs signaling via EGFR in human cancer cell lines.

    PubMed

    Ali, Imran; Damdimopoulou, Pauliina; Stenius, Ulla; Halldin, Krister

    2015-04-25

    Cadmium (Cd) is an environmental contaminant classified as carcinogenic to humans by the International Agency for Research on Cancer, supported by data from occupational exposure. Environmentally relevant dietary exposure to Cd has recently been associated with osteoporosis and cancers of the prostate, endometrium, and breast in the general population. The low exposure effects have been proposed to result from endocrine modulative properties of Cd, which mimic the physiological actions of estrogen and androgen. However, the mechanism of action of Cd is an unanswered question. We have shown previously, using mouse models, that canonical estrogen receptor signaling is not involved in estrogen mimicry effects of Cd. Instead, low-level Cd exposure stimulated the mitogen-activated protein kinases (MAPKs) ERK1/2 in these mice. Here we investigate further the ERK1/2 MAPK signaling activation by Cd in vitro by using nanomolar concentrations of cadmium chloride (CdCl2) in three different human carcinoma cell lines: HepG2, MCF-7, and ECC-1. The findings also were confirmed in previously collected mouse tissue samples. We show that 10(-8)M levels of CdCl2 activate ERK1/2 (Tyr 204) and the p53 specific ubiquitin ligase Mdm2 (Ser 166) via Raf and MEK by acting through the epidermal growth factor receptor (EGFR). Furthermore, our results suggest that the CdCl2-induced activation of ERK1/2 and Mdm2 may interfere with the p53 response to genotoxic compounds in cancer cell lines. Our data collectively suggest that nanomolar levels of CdCl2 activate Raf-MEK-ERK1/2 via EGFR. We hypothesize that this signaling cascade may be involved in observed low exposure effects of Cd in certain human populations.

  7. Resveratrol prevents cadmium activation of Erk1/2 and JNK pathways from neuronal cell death via protein phosphatases 2A and 5.

    PubMed

    Liu, Chunxiao; Zhang, Ruijie; Sun, Chenxia; Zhang, Hai; Xu, Chong; Liu, Wen; Gao, Wei; Huang, Shile; Chen, Long

    2015-11-01

    Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative disorders. Resveratrol, a natural product, has been found to exert neuroprotective effects. However, little is known regarding the effect of resveratrol on Cd-evoked neurotoxicity. Here, we show that resveratrol effectively reversed Cd-elicited cell viability reduction, morphological change, nuclear fragmentation and condensation, as well as activation of caspase-3 in neuronal cells, implying neuroprotection against Cd-poisoning by resveratrol. Further research revealed that both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases 1/2 (Erk1/2) were involved in the inhibitory effect of resveratrol on Cd-induced cell death, as selective inhibitors of Erk1/2 (U0126) and JNK (SP600125), or over-expression of dominant negative mitogen-activated protein kinase kinase 1 (MKK1) or dominant negative c-Jun potentiated resveratrol's prevention of Cd-induced phosphorylation of JNK and Erk1/2, as well as cell death in neuronal cells. Interestingly, resveratrol potently rescued the cells from Cd-induced suppression of protein phosphatases 2A (PP2A) and 5 (PP5) activity. Over-expression of PP2A or PP5 strengthened the inhibitory effects of resveratrol on Cd-induced activation of Erk1/2 and/or JNK, as well as cell death. The results indicate that resveratrol prevents Cd-induced activation of Erk1/2 and JNK pathways and neuronal cell death in part via activating PP2A and PP5. Our findings strongly support the notion that resveratrol may serve as a potential therapeutic agent in the prevention of Cd-induced neurodegenerative diseases.

  8. Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway.

    PubMed

    D'Ambrosio, Steven M; Han, Chunhua; Pan, Li; Kinghorn, A Douglas; Ding, Haiming

    2011-06-10

    Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of the EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway.

  9. Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

    PubMed Central

    D’Ambrosio, Steven M.; Han, Chunhua; Pan, Li; Kinghorn, A. Douglas; Ding, Haiming

    2011-01-01

    Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003), was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compound 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. PMID:21596018

  10. Inhibition of the Ras/Raf/ERK1/2 Signaling Pathway Restores Cultured Spinal Cord-Injured Neuronal Migration, Adhesion, and Dendritic Spine Development.

    PubMed

    Xu, Dongdong; Cao, Fujiang; Sun, Shiwei; Liu, Tao; Feng, Shiqing

    2016-08-01

    The Ras/Raf/ERK1/2 signaling pathway plays an important role in central and peripheral neurons in functions such as dendritic arborization, neuronal polarity, and axon assembly. However, emerging evidence also shows that up-regulation of this signaling pathway may lead to the development of spinal cord injury. The present study aimed to determine the effects of Ras/Raf/ERK1/2 signaling pathway inhibition on properties of spinal cord-injured neurons. First, neurons from spinal cord-injured C57BL/6 J mouse pups and sham-operated C57BL/6 J mouse pups were harvested. Then, immunofluorescence, western blotting, cell adhesion and cell migration assays, and DiI labeling were employed to investigate the effect of Ras/Raf/ERK1/2 signaling pathway inhibition on spinal cord-injured neurons. Immunofluorescence results of synapse formation indicated that the experimental spinal cord injury model was successfully established. Western blot results identified upregulated Erk phosphorylation in the spinal cord-injured neurons, and also showed that U0126 inhibited phosphorylation of Erk, which is a downstream kinase in the Ras/Raf signaling pathway. Additionally, cell migration and adhesion was significantly increased in the spinal cord-injured neurons. DiI labeling results also showed an increased formation of mature spines after inhibition of Ras/Raf/ERK1/2 signaling. Taken together, these results suggested that the Ras/Raf/ERK1/2 signaling pathway could serve as an effective treatment target for spinal cord injury.

  11. Activation of EGFR/ERBB2 via pathways involving ERK1/2, P38 MAPK, AKT and FOXO enhances recovery of diabetic hearts from ischemia-reperfusion injury.

    PubMed

    Akhtar, Saghir; Yousif, Mariam H M; Chandrasekhar, Bindu; Benter, Ibrahim F

    2012-01-01

    This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.

  12. Inhibition of VCAM-1 expression on mouse vascular smooth muscle cells by lobastin via downregulation of p38, ERK 1/2 and NF-κB signaling pathways.

    PubMed

    Lee, Kyoungran; Yim, Joung-Han; Lee, Hong-Kum; Pyo, Suhkneung

    2016-01-01

    Atherosclerosis is a chronic inflammatory disease, the progression of which is associated with the increased expression of cell adhesion molecules on vascular smooth muscle cells (VSMCs). Lobastin is a new pseudodepsidone isolated from Stereocaulon alpinum, Antarctic lichen, which is known to have antioxidant and antibacterial activities. However, the nature of the biological effects of lobastin still remains unclear. In the present study, we examine the effect of lobastin on the expression of vascular cell adhesion molecules (VCAM-1) induced by TNF-α in the cultured mouse VSMC cell line, MOVAS-1. Pretreatment of VSMCs for 2 h with lobastin (0.1-10 μg/ml) concentration-dependently inhibited TNF-α-induced protein expression of VCAM-1. Lobastin also inhibited TNF-α-induced production of intracellular reactive oxygen species (ROS). Lobastin abrogated TNF-α-induced phosphorylation of p38 and ERK 1/2, but not JNK, and also inhibited TNF-α-induced NK-κB activation. In addition, lobastin suppressed TNF-α-induced IκB kinase activation, subsequent degradation of IκBα and nuclear translocation of p65 NF-κB. Our results indicate that lobastin downregulates the TNF-α-mediated induction of VCAM-1 in VSMC by inhibiting the p38, ERK 1/2 and NF-κB signaling pathways and intracellular ROS generation. Thus, lobastin may be an important regulator of inflammation in the atherosclerotic lesion and a novel therapeutic drug for the treatment of atherosclerosis.

  13. Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model.

    PubMed

    Tabana, Yasser M; Hassan, Loiy Elsir A; Ahamed, Mohamed B Khadeer; Dahham, Saad S; Iqbal, Muhammad Adnan; Saeed, Mohammed A A; Khan, Md Shamsuddin S; Sandai, Doblin; Majid, Aman S Abdul; Oon, Chern Ein; Majid, Amin Malik S A

    2016-09-01

    We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06μM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2. PMID:27133199

  14. Epieriocalyxin A Induces Cell Apoptosis Through JNK and ERK1/2 Signaling Pathways in Colon Cancer Cells.

    PubMed

    Wang, Zhou; Xu, Zhijie; Niu, Zhengchuan; Liang, Benjia; Niu, Jun

    2015-11-01

    Colorectal cancer is one of the most commonly diagnosed cancers in the world. Currently, drug resistance of cancer cell to chemotherapy is a major cause for cancer recurrence and death of the patients; therefore, new therapeutic strategy is required to improve the care of colorectal cancer patients. The Chinese herb, Isodon eriocalyx, has been used a therapeutic for a long time in China. In this study, we showed that Epieriocalyxin A (EpiA), a diterpenoid isolated from I. eriocalyx, suppressed Caco-2 colon cancer cell growth. EpiA induced annexin V flipping in cell membrane and DNA fragment. We also showed that EpiA induced the generation of ROS in cells, as well as damage of the mitochondrial membrane. Western blot results showed that both JNK and ERK1/2 activation was decreased after EpiA treatment in a dose-dependent manner. EpiA increased the expression of caspase 3 and Bax, and decreased Bcl2 expression. Our results suggest that EpiA is a novel compound that induces colon cancer apoptosis. EpiA could be a potential drug for colon cancer therapy in the future. PMID:27352353

  15. Acquired lymphangiectasis.

    PubMed

    Celis, A V; Gaughf, C N; Sangueza, O P; Gourdin, F W

    1999-01-01

    Acquired lymphangiectasis is a dilatation of lymphatic vessels that can result as a complication of surgical intervention and radiation therapy for malignancy. Acquired lymphangiectasis shares clinical and histologic features with the congenital lesion, lymphangioma circumscriptum. Diagnosis and treatment of these vesiculo-bullous lesions is important because they may be associated with pain, chronic drainage, and cellulitis. We describe two patients who had these lesions after treatment for cancer and review the pertinent literature. Although a number of treatment options are available, we have found CO2 laser ablation particularly effective. PMID:9932832

  16. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway.

    PubMed

    Amonyingcharoen, Sumet; Suriyo, Tawit; Thiantanawat, Apinya; Watcharasit, Piyajit; Satayavivad, Jutamaad

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1-40 µM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth.

  17. TRPM6 expression and cell proliferation are up-regulated by phosphorylation of ERK1/2 in renal epithelial cells

    SciTech Connect

    Ikari, Akira Okude, Chiaki; Sawada, Hayato; Yamazaki, Yasuhiro; Sugatani, Junko; Miwa, Masao

    2008-05-16

    Transient receptor potential melastatin 6 (TRPM6) is a magnesium channel and expressed in the intestine and renal distal tubules. Little is known about the regulatory mechanism of TRPM6 expression and the role of magnesium influx. EGF increased the phosphorylation of ERK1/2 and TRPM6 expression that were inhibited by U0126 in renal epithelial NRK-52E cells. Furthermore, EGF enhanced the influx of magnesium, whereas U0126 and TRPM6 siRNA inhibited it. EGF increased the proportion of cells in S phase, whereas U0126 and TRPM6 siRNA increased the proportion in G1 phase. The phosphorylation of ERK1/2 may up-regulate TRPM6 expression and magnesium influx, resulting in an increase in cell proliferation with a shift from G1 to S phase.

  18. Chemopreventive effect of resveratrol and apocynin on pancreatic carcinogenesis via modulation of nuclear phosphorylated GSK3β and ERK1/2

    PubMed Central

    Kato, Akihisa; Naiki-Ito, Aya; Nakazawa, Takahiro; Hayashi, Kazuki; Naitoh, Itaru; Miyabe, Katsuyuki; Shimizu, Shuya; Kondo, Hiromu; Nishi, Yuji; Yoshida, Michihiro; Umemura, Shuichiro; Hori, Yasuki; Mori, Toshio; Tsutsumi, Masahiro; Kuno, Toshiya; Suzuki, Shugo; Kato, Hiroyuki; Ohara, Hirotaka; Joh, Takashi; Takahashi, Satoru

    2015-01-01

    Despite progress in clinical cancer medicine in multiple fields, the prognosis of pancreatic cancer has remained dismal. Recently, chemopreventive strategies using phytochemicals have gained considerable attention as an alternative in the management of cancer. The present study aimed to evaluate the chemopreventive effects of resveratrol (RV) and apocynin (AC) in N-Nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamster. RV- and AC-treated hamsters showed significant reduction in the incidence of pancreatic cancer with a decrease in Ki-67 labeling index in dysplastic lesions. RV and AC suppressed cell proliferation of human and hamster pancreatic cancer cells by inhibiting the G1 phase of the cell cycle with cyclin D1 downregulation and inactivation of AKT-GSK3β and ERK1/2 signaling. Further, decreased levels of GSK3βSer9 and ERK1/2 phosphorylation and cyclin D1 expression in the nuclear fraction were observed in cells treated with RV or AC. Nuclear expression of phosphorylated GSK3βSer9 was also decreased in dysplastic lesions and adenocarcinomas of hamsters treated with RV or AC in vivo. These results suggest that RV and AC reduce phosphorylated GSK3βSer9 and ERK1/2 in the nucleus, resulting in inhibition of the AKT-GSK3β and ERK1/2 signaling pathways and cell cycle arrest in vitro and in vivo. Taken together, the present study indicates that RV and AC have potential as chemopreventive agents for pancreatic cancer. PMID:26556864

  19. Endophilin-1 regulates blood-brain barrier permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway.

    PubMed

    Liu, Wenjing; Wang, Ping; Shang, Chao; Chen, Lin; Cai, Heng; Ma, Jun; Yao, Yilong; Shang, Xiuli; Xue, Yixue

    2014-07-21

    The blood-brain barrier (BBB) plays a pivotal role in maintenance and regulation of the neural microenvironment. Brain endothelial cells (BECs), held together by tight junctions (TJs), have a primary role in restricting the permeability of the BBB. Endophilin-1 is a multifunctional protein that influences epithelial growth factor receptor (EGFR) endocytosis and degradation and plays an important role in regulating the glomerular filtration barrier in the kidney. Endophilin-1 likely plays a similar role in controlling BBB permeability. In this study, we therefore analyzed the expression and function of endophilin-1 in the human BEC line hCMEC/D3. Our results show that endophilin-1 over-expression reduced the expression of the TJ-associated proteins ZO-1 and occludin and increased the paracellular permeability of hCMEC/D3 cells, whereas silencing of endogenous endophilin-1 yielded the opposite results. Over-expression of ZO-1 and occludin prevented the increase in permeability induced by endophilin-1 over-expression, whereas down-regulation of ZO-1 and occludin prevented the reduction in permeability induced by endophilin-1 silencing. Co-localization and co-immunoprecipitation experiments suggested that endophilin-1 interacts with the EGFR. The levels of EGFR and its downstream effector phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) are significantly decreased when endophilin-1 is over-expressed. Conversely, endophilin-1 down-regulation led to markedly increased levels of these proteins. In addition, the reduced permeability induced by endophilin-1 down-regulation was blocked by AG1478 and PD98059, inhibitors of EGFR and ERK1/2, respectively. Up-regulation of ZO-1 and occludin was blocked by the EGFR and ERK1/2 inhibitors. These results suggest that endophilin-1 regulates BBB permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway in BECs.

  20. Changes in the expression of extracellular regulated kinase (ERK 1/2) in the R6/2 mouse model of Huntington's disease after phosphodiesterase IV inhibition.

    PubMed

    Fusco, Francesca R; Anzilotti, Serenella; Giampà, Carmela; Dato, Clemente; Laurenti, Daunia; Leuti, Alessandro; Colucci D'Amato, Luca; Perrone, Lorena; Bernardi, Giorgio; Melone, Mariarosa A B

    2012-04-01

    The mitogen-activated protein kinases (MAPKs) superfamily comprises three major signaling pathways: the extracellular signal-regulated protein kinases (ERKs), the c-Jun N-terminal kinases or stress-activated protein kinases (JNKs/SAPKs) and the p38 family of kinases. ERK 1/2 signaling has been implicated in a number of neurodegenerative disorders, including Huntington's disease (HD). Phosphorylation patterns of ERK 1/2 and JNK are altered in cell models of HD. In this study, we aimed at studying the correlations between ERK 1/2 and the neuronal vulnerability to HD degeneration in the R6/2 transgenic mouse model of HD. Single and double-label immunofluorescence for phospho-ERK (pERK, the activated form of ERK) and for each of the striatal neuronal markers were employed on perfusion-fixed brain sections from R6/2 and wild-type mice. Moreover, Phosphodiesterase 4 inhibition through rolipram was used to study the effects on pERK expression in the different types of striatal neurons. We completed our study with western blot analysis. Our study shows that pERK levels increase with age in the medium spiny striatal neurons and in the parvalbumin interneurons, and that rolipram counteracts such increase in pERK. Conversely, cholinergic and somatostatinergic interneurons of the striatum contain higher levels of pERK in the R6/2 mice compared to the controls. Rolipram induces an increase in pERK expression in these interneurons. Thus, our study confirms and extends the concept that the expression of phosphorylated ERK 1/2 is related to neuronal vulnerability and is implicated in the pathophysiology of cell death in HD.

  1. Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

    PubMed

    Damm, Ellen; Buech, Thomas R H; Gudermann, Thomas; Breit, Andreas

    2012-04-01

    α-Melanocyte-stimulating hormone (α-MSH)-induced activation of the melanocortin-4 receptor in hypothalamic neurons increases energy expenditure and inhibits food intake. Active hypothalamic AMP-activated protein kinase (AMPK) has recently been reported to enhance food intake, and in vivo experiments suggested that intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of AMPK activity. However, it is not clear whether α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Here, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK phosphorylation at Thr(172), which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr(172) and consequently decreased phosphorylation of the established AMPK substrate acetyl-coenzyme A-carboxylase at Ser(79). Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein kinase A (PKA) or ERK-1/2, pointing to an important role of both kinases in this process. Because α-MSH-induced activation of ERK-1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1, but not inhibition of calcium-calmodulin-dependent kinase kinase-β or TGFβ-activated kinase-1 decreased basal phosphorylation of AMPK and its dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK activity via a linear pathway, including PKA, ERK-1/2, and liver kinase B-1 in GT1-7 cells. Given the importance of the melanocortin system in the formation of adipositas, detailed knowledge about this pathway might help to develop drugs targeting obesity.

  2. Expression of tumor necrosis factor-α-induced protein 8 in stage III gastric cancer and the correlation with DcR3 and ERK1/2

    PubMed Central

    HU, RUYI; LIU, WENMING; QIU, XINGFENG; LIN, ZHENGHE; XIE, YAN; HONG, XINGYA; PAERHATI, REYILA; QI, ZHONGQUAN; ZHUANG, GUOHONG; LIU, ZHONGCHEN

    2016-01-01

    Tumor necrosis factor (TNF)-α-induced protein 8 (TIPE) is a recently identified protein that is considered to be associated with various malignancies, including esophageal, breast and pancreatic cancer; however, the importance of TIPE in gastric cancer (GC) remains unknown. Decoy receptor 3 (DcR3) is a member of the tumor necrosis factor receptor superfamily that is expressed in digestive system neoplasms. The expression of DcR3 is regulated by the mitogen-activated protein kinase (MAPK)/MAPK kinase/extracellular signal-regulated kinase (ERK) signaling pathway. Reverse transcription-polymerase chain reaction was performed to detect the expression of TIPE, ERK and DcR3 in the pathological and tumor-adjacent normal gastric tissues of 30 patients that demonstrated stage III gastric adenocarcinoma. The expression and distribution of the TIPE protein was examined using immunohistochemistry, and the clinical significance and expression levels of DcR3 and ERK1/2 were evaluated. The expression of TIPE, ERK1/2 and DcR3 in the tumor tissues of GC was significantly increased compared with paracarcinoma tissues (P<0.05). In addition, TIPE expression positively correlated with DcR3 and ERK1 levels (r=0.538 and r=0.462, respectively; P<0.05). There was no statistical difference between tumor tissues from patients with varying age, gender, differentiation or lymph node metastasis (P>0.05). TIPE may be vital in the progression of GC. TIPE may be associated with the expression of DcR3 and ERK1/2, which may be involved in the cell apoptosis of GC. The present study elucidates the potential function of TIPE as a novel marker and therapeutic target for GC. PMID:26998086

  3. CyPA-CD147-ERK1/2-cyclin D2 signaling pathway is upregulated during rat left ventricular hypertrophy.

    PubMed

    Tang, Fu-Cai; Wang, Hong-Yan; Ma, Ming-Ming; Guan, Tian-Wang; Pan, Long; Yao, Dun-Chen; Chen, Ya-Lan; Chen, Wei-Bei; Tu, Yong-Sheng; Fu, Xiao-Dong

    2015-08-25

    The changes of serum cyclophilin A (CyPA), its receptor CD147 and the downstream signaling pathway during the process of cardiac hypertrophy remain unknown. The present study aims to investigate the relationships between CyPA-CD147-ERK1/2-cyclin D2 signaling pathway and the development of cardiac hypertrophy. Left ventricular hypertrophy was prepared by 2-kidney, 2-clip in Sprague-Dawley rats and observed for 1 week, 4 and 8 weeks. Left ventricular hypertrophy was evaluated by ratio of left ventricular heart weight to body weight (LVW/BW) and cardiomyocyte cross sectional area (CSA). CyPA levels in serum were determined with a rat CyPA ELISA kit. Expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular myocytes were determined by Western blot and immunostaining. Compared with sham groups, systolic blood pressure reached hypertensive levels at 4 weeks in 2K2C groups. LVW/BW and CSA in 2K2C groups were significantly increased at 4 and 8 weeks after clipping. ELISA results indicated a prominent increase in serum CyPA level associated with the degree of left ventricular hypertrophy. Western blot revealed that the expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular tissues were also remarkably increased as the cardiac hypertrophy developed. The results of the present study demonstrates that serum CyPA and CyPA-CD147-ERK1/2-cyclin D2 signaling pathway in ventricular tissues are time-dependently upregulated and activated with the process of left ventricular hypertrophy. These data suggest that CyPA-CD147 signaling cascade might play a role in the pathogenesis of left ventricular hypertrophy, and CyPA might be a prognosticator of the degree of left ventricular hypertrophy. PMID:26300251

  4. Apelin-13 promotes cardiomyocyte hypertrophy via PI3K-Akt-ERK1/2-p70S6K and PI3K-induced autophagy.

    PubMed

    Xie, Feng; Liu, Wei; Feng, Fen; Li, Xin; He, Lu; Lv, Deguan; Qin, Xuping; Li, Lifang; Li, Lanfang; Chen, Linxi

    2015-12-01

    Apelin is highly expressed in rat left ventricular hypertrophy Sprague Dawley rat models, and it plays a crucial role in the cardiovascular system. The aim this study was to clarify whether apelin-13 promotes hypertrophy in H9c2 rat cardiomyocytes and to investigate its underlying mechanism. The cardiomyocyte hypertrophy was observed by measuring the diameter, volume, and protein content of H9c2 cells. The activation of autophagy was evaluated by observing the morphology of autophagosomes by transmission electron microscopy, observing the subcellular localization of LC3 by light microscopy, and detecting the membrane-associated form of LC3 by western blot analysis. The phosphatidylinositol 3-kinase (PI3K) signaling pathway was identified and the proteins expression was detected using western blot analysis. The results revealed that apelin-13 increased the diameter, volume, and protein content of H9c2 cells and promoted the phosphorylation of PI3K, Akt, ERK1/2, and p70S6K. Apelin-13 activated the PI3K-Akt-ERK1/2-p70S6K pathway. PI3K inhibitor LY294002, Akt inhibitor 1701-1, ERK1/2 inhibitor PD98059 attenuated the increase of the cell diameter, volume, protein content induced by apelin-13. Apelin-13 increased the autophagosomes and up-regulated the expressions of beclin 1 and LC3-II/I both transiently and stably. The autophagy inhibitor 3MA ameliorated the increase of cell diameter, volume, and protein content that were induced by apelin-13. These results suggested that apelin-13 promotes H9c2 rat cardiomyocyte hypertrophy via PI3K-Akt-ERK1/2-p70S6K and PI3K-induced autophagy. PMID:26607438

  5. CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

    SciTech Connect

    Chen, Tian Jun; Gao, Fei; Yang, Tian; Thakur, Asmitanand; Ren, Hui; Li, Yang; Zhang, Shuo; Wang, Ting; Chen, Ming Wei

    2013-07-19

    Highlights: •CDK-associated Cullin 1 (CAC1) expression increases in human lung carcinoma. •CAC1 promotes the proliferation of lung cancer A549 cells. •CAC1 promotes human lung cancer A549 cell proliferation with activation of ERK1/2. -- Abstract: Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer.

  6. Targeting Transforming Growth Factor-Beta1 (TGF-β1) Inhibits Tumorigenesis of Anaplastic Thyroid Carcinoma Cells Through ERK1/2-NFκkB-PUMA Signaling.

    PubMed

    Yin, Qiang; Liu, Shan; Dong, Anbing; Mi, Xiufang; Hao, Fengyun; Zhang, Kejun

    2016-06-30

    BACKGROUND The transforming growth factor-beta (TGF-β) signaling pathway plays a critical role in promoting tumor growth. TGF-β1was found to be overexpressed in anaplastic thyroid cancer (ATC). We therefore tested our hypothesis that targeting TGF-β1 inhibits tumorigenesis of ATC cells. MATERIAL AND METHODS Effects of TGF-β1 stimulation or TGF-β1 inhibition by small interfering RNA (TGF-β1siRNA) on proliferation, colony formation, and apoptosis in 8505C cells in vitro was detected using siRNAs and inhibitors to examine the TGF-β1 signaling pathway. A subcutaneously implanted tumor model of 8505C cells in nude mice was used to assess the effects of TGF-β1 inhibition on tumorigenesis development. RESULTS TGF-β1siRNAs decreased proliferation and colony formation, and increased apoptosis in 8505C cells in vitro and inhibited tumor growth in vivo. TGF-β1siRNA inhibited phosphorylation ERK1/2 (pERK1/2) and increased p65-dependant PUMA mRNA and protein expression. Knockdown of p65 or PUMA by siRNA reduced TGF-β1siRNA-induced apoptosis, as well as caspase-3 and PARP activation. Upregulation of p65 or PUMA expression by TGF-β1siRNA requires pERK1/2 inhibition. TGF-β1 shRNA inhibited tumor growth in vivo. CONCLUSIONS Therapies targeting the TGF-β1 pathway may be more effective to prevent primary tumor formation. The ability of this therapy to decrease tumorigenesis may be related to ERK1/2/NF-κB/PUMA signaling.

  7. Involvement of Raf-1/MEK/ERK1/2 signaling pathway in zinc-induced injury in rat renal cortical slices.

    PubMed

    Kohda, Yuka; Matsunaga, Yoshiko; Shiota, Ryugo; Satoh, Tomohiko; Kishi, Yuko; Kawai, Yoshiko; Gemba, Munekazu

    2006-08-01

    Zinc is an essential nutrient that can also be toxic. We have previously reported that zinc-related renal toxicity is due, in part, to free radical generation in the renal epithelial cell line, LLC-PK(1) cells. We have also shown that an MEK1/2 inhibitor, U0126, markedly inhibits zinc-induced renal cell injury. In this study, we investigated the role of an upstream MEK/ERK pathway, Raf-1 kinase pathway, and the transcription factor and ERK substrate Elk-1, in rat renal cortical slices exposed to zinc. Immediately after preparing slices from rat renal cortex, the slices were incubated in medium containing Raf-1 and MEK inhibitors. ERK1/2 and Elk-1 activation were determined by Western blot analysis for phosphorylated ERK (pERK) 1/2 and phosphorylated Elk-1 (pElk-1) in nuclear fractions prepared from slices exposed to zinc. Zinc caused not only increases in 4-hydroxynonenal (4-HNE) modified protein and lipid peroxidation, as an index of oxidant stress, and decreases in PAH accumulation, as that of renal cell injury in the slices. Zinc also induced a rapid increase in ERK/Elk-1 activity accompanied by increased expressions of pERK and pElk-1 in the nuclear fraction. A Raf-1 kinase inhibitor and an MEK1/2 inhibitor U0126 significantly attenuated zinc-induced decreases PAH accumulation in the slices. The Raf-1 kinase inhibitor and U0126 also suppressed ERK1/2 activation in nuclear fractions prepared from slices treated with zinc. The present results suggest that a Raf-1/MEK/ERK1/2 pathway and the ERK substrate Elk-1 are involved in free radical-induced injury in rat renal cortical slices exposed to zinc.

  8. Mannose-capped Lipoarabinomannan from Mycobacterium tuberculosis induces IL-37 production via upregulating ERK1/2 and p38 in human type II alveolar epithelial cells.

    PubMed

    Huang, Zhen; Zhao, Gao Wei; Gao, Chun Hai; Chi, Xiu Wen; Zeng, Tao; Hu, Yan Wei; Zheng, Lei; Wang, Qian

    2015-01-01

    The major surface lipoglycan of Mycobacterium tuberculosis (M. tb), mannose-capped lipoarabinomannan (ManLAM), is an immunosuppressive epitope of M. tb. Interleukin (IL)-37, is a newly identified anti-inflammatory cytokine, which reduces systemic and local inflammation. However, the correlation between ManLAM and IL-37 remains unknown. Therefore, in this study, we investigate the possible role and relative molecular mechanism of ManLAM in IL-37 production of human type II alveolar epithelial cells by using A549 cell line. Here, we report that M. tb induced IL-37 mRNA and protein expression in a time-dependent manner. We next fractionated components of M. tb using chloroform: methanol (C:M) and water. In sharp contrast to the C:M phase, water phase was mainly responsible for the production of IL-37. Since ManLAM is the major component of water phase, we found that ManLAM induced IL-37 mRNA and protein expression in a time and dose-dependent manner, while this activity was almost totally abolished by the ERK1/2 (U0126) and p38 (SB203580) inhibitor. ManLAM stimulation significantly induced ERK1/2 and p38 phosphorylation in A549 cells, as well as cell surface TLR2 expression. After interfering TLR2 expression, ERK1/2 and p38 phosphorylation levels were markedly decreased, and also IL-37 production. Though ManLAM also promoted TLR4 expression on A549 cells, TLR4 interference showed no influence on ManLAM-induced IL-37 production. Our results indicate that ManLAM induces IL-37 production in human type II alveolar epithelial cells via up-regulating TLR2/p38 or ERK1/2 pathway, and this provide an important evidence to explain the pathological role of ManLAM that contribute to the persistence of M. tb. PMID:26221267

  9. Targeting Transforming Growth Factor-Beta1 (TGF-β1) Inhibits Tumorigenesis of Anaplastic Thyroid Carcinoma Cells Through ERK1/2-NFκkB-PUMA Signaling.

    PubMed

    Yin, Qiang; Liu, Shan; Dong, Anbing; Mi, Xiufang; Hao, Fengyun; Zhang, Kejun

    2016-01-01

    BACKGROUND The transforming growth factor-beta (TGF-β) signaling pathway plays a critical role in promoting tumor growth. TGF-β1was found to be overexpressed in anaplastic thyroid cancer (ATC). We therefore tested our hypothesis that targeting TGF-β1 inhibits tumorigenesis of ATC cells. MATERIAL AND METHODS Effects of TGF-β1 stimulation or TGF-β1 inhibition by small interfering RNA (TGF-β1siRNA) on proliferation, colony formation, and apoptosis in 8505C cells in vitro was detected using siRNAs and inhibitors to examine the TGF-β1 signaling pathway. A subcutaneously implanted tumor model of 8505C cells in nude mice was used to assess the effects of TGF-β1 inhibition on tumorigenesis development. RESULTS TGF-β1siRNAs decreased proliferation and colony formation, and increased apoptosis in 8505C cells in vitro and inhibited tumor growth in vivo. TGF-β1siRNA inhibited phosphorylation ERK1/2 (pERK1/2) and increased p65-dependant PUMA mRNA and protein expression. Knockdown of p65 or PUMA by siRNA reduced TGF-β1siRNA-induced apoptosis, as well as caspase-3 and PARP activation. Upregulation of p65 or PUMA expression by TGF-β1siRNA requires pERK1/2 inhibition. TGF-β1 shRNA inhibited tumor growth in vivo. CONCLUSIONS Therapies targeting the TGF-β1 pathway may be more effective to prevent primary tumor formation. The ability of this therapy to decrease tumorigenesis may be related to ERK1/2/NF-κB/PUMA signaling. PMID:27356491

  10. Role of the ERK1/2 Signaling Pathway in Osteogenesis of Rat Tendon-Derived Stem Cells in Normoxic and Hypoxic Cultures

    PubMed Central

    Li, Pei; Xu, Yuan; Gan, Yibo; Song, Lei; Zhang, Chengmin; Wang, Liyuan; Zhou, Qiang

    2016-01-01

    Background: Ectopic ossification and increased vascularization are two common phenomena in the chronic tendinopathic tendon. The increased vascularization usually leads to an elevated local oxygen tension which is one of micro-environments that can influence differentiate status of stem cells. Objective: This study aimed to investigate the osteogenesis capacity of rat tendon-derived stem cells TDSCs (rTDSCs) in normoxic and hypoxic cultures, and to study the role of ERK1/2 signaling pathway in this process. Methods: rTDSCs were subjected to osteogenesis inductive culture in hypoxic (3% O2) and normoxic (20% O2) conditions. The inhibitor U0126 was added along with culture medium to determine the role of ERK1/2 signaling pathway. Cell viability, cell proliferation, alizarin red staining, alkaline phosphatase (AKP) activity, gene expression (ALP, osteocalcin, collagen I and RUNX2) and protein expression (p-ERK1/2 and RUNX2) of osteogenic-cultured rTSDCs were analyzed in this study. Results: Hypoxic and normoxic culture had no effects on cell viability of rTDSCs, whereas the proliferation potential of rTDSCs was significantly increased in hypoxic culture. The osteogenesis capacity of rTDSCs in normoxic culture was significantly promoted compared with hypoxic culture, which was reflected by an increased alizarin red staining intensity, an elevated ALP activity, and the up-regulated gene (ALP, osteocalcin, collagen I and RUNX2) or protein (RUNX2) expression of osteogenic makers. However, the osteogenesis capacity of rTDSCs in both hypoxic and normoxic cultures was attenuated by the inhibitor U0126. Conclusion: Normoxic culture promotes osteogenic differentiation of rTDSCs compared with the hypoxic culture, and the ERK1/2 signaling pathway is involved in this process. PMID:27499695

  11. Tissue Kallikrein Alleviates Cerebral Ischemia-Reperfusion Injury by Activating the B2R-ERK1/2-CREB-Bcl-2 Signaling Pathway in Diabetic Rats

    PubMed Central

    Yuan, Kunxiong; Hu, Bin; Sang, Hongfei; Xie, Yi; Xu, Lili; Cao, Qinqin; Chen, Xin; Zhao, Lingling; Liu, Xinfeng; Liu, Ling; Zhang, Renliang

    2016-01-01

    Diabetes mellitus (DM) substantially increases the risk of ischemic stroke and reduces the tolerance to ischemic insults. Tissue kallikrein (TK) has been demonstrated to protect neurons from ischemia/reperfusion (I/R) injury in orthoglycemic model by activating the bradykinin B2 receptor (B2R). Considering the differential effects of B2R or bradykinin B1 receptor (B1R) on cardioprotection and neuroprotection in I/R with or without diabetes, this study was designed to investigate the role of TK during cerebral I/R injury in streptozotocin-induced diabetic rats. Intravenous injection of TK inhibited apoptosis in neurons, alleviated edema and inflammatory reactions after focal cerebral I/R, significantly reduced the infarct volume, and improved functional recovery. These beneficial effects were accompanied by activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP response element-binding (CREB), and Bcl-2 signal proteins. Inhibition of the B2R or ERK1/2 pathway abated the effects of TK, whereas an antagonist of B1R enhanced the effects. These findings reveal that the neuroprotective effect of TK against cerebral I/R injury in streptozotocin-induced diabetic rats mainly involves the enhancement of B2R and ERK1/2-CREB-Bcl-2 signaling pathway activity. PMID:27446506

  12. Inhibitory effect of puerarin on vascular smooth muscle cells proliferation induced by oxidised low-density lipoprotein via suppressing ERK 1/2 phosphorylation and PCNA expression.

    PubMed

    Hu, Yanwu; Liu, Kai; Bo, Sun; Yan, Mengtong; Zhang, Yang; Miao, Chunsheng; Ren, Liqun

    2016-02-01

    Puerarin, an isoflavonoid isolated from the traditional Chinese herbal medicine Pueraria lobata (Wild.) Ohwi, has been shown to process antioxidant, anti-inflammatory, anti-cancer, anti-hypercholesterolemic, and anti-hyperglycemic activities in vivo and in vitro. The aim of the present study was to investigate the antiproliferative effects and the possible mechanisms of puerarin in vascular smooth muscle cells (VSMCs) stimulated with oxidised low-density lipoprotein (ox-LDL). VSMCs were cultured and pretreated with different concentrations of puerarin (0, 1, 10, 50 µM) before stimulated by ox-LDL (50 µg/mL). Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of puerarin on cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 were detected by western blotting analysis. The results indicated that puerarin significantly inhibited VSMCs proliferation induced by ox-LDL and phosphorylation of ERK 1/2. Furthermore, puerarin also blocked the ox-LDL-induced cell-cycle progression at G1/S-interphase and down-regulated the expression of PCNA of VSMCs. The results suggest puerarin inhibits ox-LDL-induced proliferation of VSMCs by suppressing ERK 1/2 phosphorylation and PCNA expression.

  13. RKIP phosphorylation-dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3.

    PubMed

    Hahm, Jong Ryeal; Ahmed, Mahmoud; Kim, Deok Ryong

    2016-09-01

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-l-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-l-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity. PMID:27470585

  14. Dexamethasone in the presence of desipramine enhances MAPK/ERK1/2 signaling possibly via its interference with β-arrestin.

    PubMed

    Lucki, Anat; Klein, Ehud; Karry, Rachel; Ben-Shachar, Dorit

    2014-01-01

    Antidepressant medication is the standard treatment for major depression disorder (MDD). However, the response to these treatments is often incomplete and many patients remain refractory. In the present study, we show that the glucocorticoid receptor (GR) agonist dexamethasone (DEX) increased MAPK/ERK1/2 signaling in the presence of the noradrenergic antidepressant, desipramine (DMI), while no such effect was induced by DEX or DMI alone in human neuroblastoma SH-SY5Y cells. This enhancement was dependent on the activation of both α(2) adrenergic receptors (AR) and GR. The timing of MAPK/ERK1/2 activation as well as DEX-induced reduction in membranous α(2) AR suggests the involvement of a β-arrestin-dependent mechanism. In line with the latter, DEX increased cytosolic and decreased membranous levels of β-arrestin. Concomitantly, DEX induced a time-dependent increase in cytosolic α(2) AR-β-arrestin interaction and a decrease in β-arrestin interaction with Mdm2 E3 ubiquitin ligase. All of these effects of DEX were prevented by the GR antagonist RU486. Our data suggest an additional intracellular role for DEX, in which activation of GR interferes with the trafficking and degradation of β-arrestin-α2c-AR complex. We suggest that such an interaction in the presence of DMI can enhance MAPK/ERK1/2 signaling, a key player in neural plasticity and neurogenesis processes, which is impaired in MDD, while stimulated by antidepressants.

  15. Involvement of ERK1/2 pathway in neuroprotective effects of pyrroloquinoline quinine against rotenone-induced SH-SY5Y cell injury.

    PubMed

    Zhang, Q; Zhang, J; Jiang, C; Qin, J; Ke, K; Ding, F

    2014-06-13

    Pyrroloquinoline quinone (PQQ), a redox cofactor in the mitochondrial respiratory chain, has been shown to protect neurons against glutamate-induced damage both in vitro and in vivo. In this study, specific inhibitors to each of the mitochondrial complexes were used to find out which reactive oxygen species (ROS)-generating sites could be affected by PQQ. Then we established an in vitro model of Parkinson's disease (PD) by exposing cultured SH-SY5Y dopaminergic cells to rotenone, a complex I inhibitor. The neuroprotective effects of PQQ were observed by pretreatment of SH-SY5Y cells with PQQ before rotenone injury, and the possible involvement of certain signaling pathways were investigated. PQQ pretreatment prevented SH-SY5Y cells from rotenone-induced apoptosis in a concentration-dependent manner. PQQ neuroprotection was associated with inhibition of intracellular ROS production, modulation of the expression of apoptosis-related Bcl-2 and Bax, and regulation of the level of superoxide dismutase, glutathione, and malondialdehyde. Meanwhile, PQQ up-regulated the gene expression of Ndufs 1, 2, and 4 (complex I subunits), and increased mitochondrial viability and mitochondrial DNA content. Furthermore, PQQ pretreatment activated ERK1/2 phosphorylation in rotenone-injured SH-SY5Y cells, while ERK1/2 inhibition suppressed PQQ neuroprotection. All the results suggested that PQQ could protect SH-SY5Y cells against rotenone injury by reducing ROS production and maintaining mitochondrial functions through activation of ERK1/2 pathway.

  16. Inhibitory effect of puerarin on vascular smooth muscle cells proliferation induced by oxidised low-density lipoprotein via suppressing ERK 1/2 phosphorylation and PCNA expression.

    PubMed

    Hu, Yanwu; Liu, Kai; Bo, Sun; Yan, Mengtong; Zhang, Yang; Miao, Chunsheng; Ren, Liqun

    2016-02-01

    Puerarin, an isoflavonoid isolated from the traditional Chinese herbal medicine Pueraria lobata (Wild.) Ohwi, has been shown to process antioxidant, anti-inflammatory, anti-cancer, anti-hypercholesterolemic, and anti-hyperglycemic activities in vivo and in vitro. The aim of the present study was to investigate the antiproliferative effects and the possible mechanisms of puerarin in vascular smooth muscle cells (VSMCs) stimulated with oxidised low-density lipoprotein (ox-LDL). VSMCs were cultured and pretreated with different concentrations of puerarin (0, 1, 10, 50 µM) before stimulated by ox-LDL (50 µg/mL). Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of puerarin on cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 were detected by western blotting analysis. The results indicated that puerarin significantly inhibited VSMCs proliferation induced by ox-LDL and phosphorylation of ERK 1/2. Furthermore, puerarin also blocked the ox-LDL-induced cell-cycle progression at G1/S-interphase and down-regulated the expression of PCNA of VSMCs. The results suggest puerarin inhibits ox-LDL-induced proliferation of VSMCs by suppressing ERK 1/2 phosphorylation and PCNA expression. PMID:27004373

  17. Tissue Kallikrein Alleviates Cerebral Ischemia-Reperfusion Injury by Activating the B2R-ERK1/2-CREB-Bcl-2 Signaling Pathway in Diabetic Rats

    PubMed Central

    Yuan, Kunxiong; Hu, Bin; Sang, Hongfei; Xie, Yi; Xu, Lili; Cao, Qinqin; Chen, Xin; Zhao, Lingling; Liu, Xinfeng; Liu, Ling; Zhang, Renliang

    2016-01-01

    Diabetes mellitus (DM) substantially increases the risk of ischemic stroke and reduces the tolerance to ischemic insults. Tissue kallikrein (TK) has been demonstrated to protect neurons from ischemia/reperfusion (I/R) injury in orthoglycemic model by activating the bradykinin B2 receptor (B2R). Considering the differential effects of B2R or bradykinin B1 receptor (B1R) on cardioprotection and neuroprotection in I/R with or without diabetes, this study was designed to investigate the role of TK during cerebral I/R injury in streptozotocin-induced diabetic rats. Intravenous injection of TK inhibited apoptosis in neurons, alleviated edema and inflammatory reactions after focal cerebral I/R, significantly reduced the infarct volume, and improved functional recovery. These beneficial effects were accompanied by activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP response element-binding (CREB), and Bcl-2 signal proteins. Inhibition of the B2R or ERK1/2 pathway abated the effects of TK, whereas an antagonist of B1R enhanced the effects. These findings reveal that the neuroprotective effect of TK against cerebral I/R injury in streptozotocin-induced diabetic rats mainly involves the enhancement of B2R and ERK1/2-CREB-Bcl-2 signaling pathway activity.

  18. RKIP phosphorylation-dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3.

    PubMed

    Hahm, Jong Ryeal; Ahmed, Mahmoud; Kim, Deok Ryong

    2016-09-01

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-l-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-l-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity.

  19. Lignosus rhinocerotis (Cooke) Ryvarden mimics the neuritogenic activity of nerve growth factor via MEK/ERK1/2 signaling pathway in PC-12 cells

    PubMed Central

    Seow, Syntyche Ling-Sing; Eik, Lee-Fang; Naidu, Murali; David, Pamela; Wong, Kah-Hui; Sabaratnam, Vikineswary

    2015-01-01

    The traditional application of the sclerotium of Lignosus rhinocerotis (tiger’s milk mushroom) by the indigenous folks as tonic and remedy to treat a variety of ailments has been documented in Malaysia. Indigenous communities claimed to have consumed the decoction to boost their alertness during hunting. Mental alertness is believed to be related to neuronal health and neuroactivity. In the present study, the cell viability and neuritogenic effects of L. rhinocerotis sclerotium hot aqueous and ethanolic extracts, and crude polysaccharides on rat pheochromocytoma (PC-12) cells were studied. Interestingly, the hot aqueous extract exhibited neuritogenic activity comparable to NGF in PC-12 cells. However, the extracts and crude polysaccharides stimulated neuritogenesis without stimulating the production of NGF in PC-12 cells. The involvements of the TrkA receptor and MEK/ERK1/2 pathway in hot aqueous extract-stimulated neuritogenesis were examined by Trk (K252a) and MEK/ERK1/2 (U0126 and PD98059) inhibitors. There was no significant difference in protein expression in NGF- and hot aqueous extract-treated cells for both total and phosphorylated p44/42 MAPK. The neuritogenic activity in PC-12 cells stimulated by hot aqueous and ethanolic extracts, and crude polysaccharides of L. rhinocerotis sclerotium mimicking NGF activity via the MEK/ERK1/2 signaling pathway is reported for the first time. PMID:26542212

  20. α-Solanine inhibits vascular endothelial growth factor expression by down-regulating the ERK1/2-HIF-1α and STAT3 signaling pathways.

    PubMed

    Wen, Zhengde; Huang, Chaohao; Xu, Yaya; Xiao, Yuwu; Tang, Lili; Dai, Juji; Sun, Hongwei; Chen, Bicheng; Zhou, Mengtao

    2016-01-15

    In tumors, vascular endothelial growth factor (VEGF) contributes to angiogenesis, vascular permeability, and tumorigenesis. In our previous study, we found that α-solanine, which is widespread in solanaceae, has a strong anti-cancer effect under normoxia. However, it is unknown whether α-solanine has a similar effect under hypoxia. We used cobalt chloride (CoCl2) to mimic hypoxia in vitro. HIF-1α, which is almost undetectable under normoxia, was significantly increased. Simultaneously, another regulator of VEGF, STAT3, was also significantly activated by CoCl2. We utilized α-solanine in co-culture with CoCl2. α-solanine decreased the expression of VEGF and loss of E-cadherin. α-solanine also suppressed the activation of phospho-ERK1/2 (p-ERK1/2), HIF-1α, and STAT3 signaling. The results provide new evidence that α-solanine has a strong anti-cancer effect via the ERK1/2-HIF-1α and STAT3 signaling pathways and suggest that it may be a potential new drug.

  1. Oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling

    PubMed Central

    Mu, Da-Wei; Guo, He-Qing; Zhou, Gao-Biao; Li, Jian-Ye; Su, Bin

    2015-01-01

    Oleanolic acid has significant pharmacological activities, such as anti-tumor, regulating blood sugar level and liver protection, which are more effective compared with free aglyconeoleanolic acid. However, it is still unknown if oleanolic acid affects the proliferation of human bladder cancer. We utilized T24 cells to study the effect of oleanolic acid on the proliferation and apoptosis of human bladder cancer. In this study, we found that the anti-cancer effect of oleanolic acid significantly suppressed cell proliferation and increased apoptosis and caspase-3 activity of T24 cells. Furthermore, Akt, mTOR and S6K protein expression was greatly inhibited in T24 cells under oleanolic acid treatment. Meanwhile, ERK1/2 of phosphorylation protein expression was significantly promoted by oleanolic acid treatment. Taken together, we provided evidences that oleanolic acid was Akt/mTOR/S6K and ERK1/2 signaling-targeting anti-tumor agent. These findings represent new evidences that oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling, and oleanolic acid may be used to prevent human bladder cancer. PMID:26823699

  2. The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.

    PubMed

    Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo

    2016-09-01

    Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells. PMID:27424123

  3. The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.

    PubMed

    Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo

    2016-09-01

    Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells.

  4. Acquired hyperpigmentations*

    PubMed Central

    Cestari, Tania Ferreira; Dantas, Lia Pinheiro; Boza, Juliana Catucci

    2014-01-01

    Cutaneous hyperpigmentations are frequent complaints, motivating around 8.5% of all dermatological consultations in our country. They can be congenital, with different patterns of inheritance, or acquired in consequence of skin problems, systemic diseases or secondary to environmental factors. The vast majority of them are linked to alterations on the pigment melanin, induced by different mechanisms. This review will focus on the major acquired hyperpigmentations associated with increased melanin, reviewing their mechanisms of action and possible preventive measures. Particularly prominent aspects of diagnosis and therapy will be emphasized, with focus on melasma, post-inflammatory hyperpigmentation, periorbital pigmentation, dermatosis papulosa nigra, phytophotodermatoses, flagellate dermatosis, erythema dyschromicum perstans, cervical poikiloderma (Poikiloderma of Civatte), acanthosis nigricans, cutaneous amyloidosis and reticulated confluent dermatitis PMID:24626644

  5. Differential Activation of Mitogen-Activated Protein Kinases, ERK 1/2, p38(MAPK) and JNK p54/p46 During Postnatal Development of Rat Hippocampus.

    PubMed

    Costa, Ana Paula; Lopes, Mark William; Rieger, Débora K; Barbosa, Sabrina Giovana Rocha; Gonçalves, Filipe Marques; Xikota, João Carlos; Walz, Roger; Leal, Rodrigo B

    2016-05-01

    Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38(MAPK), ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38(MAPK) at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38(MAPK) during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38(MAPK) are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus.

  6. Suppression of A549 cell proliferation and metastasis by calycosin via inhibition of the PKC-α/ERK1/2 pathway: An in vitro investigation

    PubMed Central

    CHENG, XU-DONG; GU, JUN-FEI; YUAN, JIA-RUI; FENG, LIANG; JIA, XIAO-BIN

    2015-01-01

    The migration and invasion of lung cancer cells into the extracellular matrix contributes to the high mortality rates of lung cancer. The protein kinase C (PKC) and downstream signaling pathways are important in the invasion and migration of lung cancer cells. Calycosin (Cal), an effector chemical from Astragalus has been reported to affect the recurrence and metastasis of cancer cells via the regulation of the protein expression of matrix metalloproteinases (MMPs). The inhibition of Cal on the migration and invasion of A549 cells was investigated in the present study. Cell viability and apoptosis assays were performed using MTT and flow cytometric analyses. A wound healing assay and Transwell invasion assay were performed to evaluate the effect of Cal on A549 cell migration and invasion. Invasion-associated proteins, including MMP-2, MMP-9, E-cadherin (E-cad), integrin β1, PKC-α and extracellular signal-regulated kinase 1/2 (ERK1/2) were detected using western blotting. In addition, PKC-α inhibitor, AEB071, and ERK1/2 inhibitor, PD98059, were used to determine the association between the suppression of PKC-α/ERK1/2 and invasion, MMP-2, MMP-9, E-cad and integrin β1. Cal was observed to suppress cell proliferation and induce apoptosis. There were significant differences between the phorbol-12-myristate-13-acetate (TPA)-induced A549 cells treated with Cal and the untreated cells in the rates of migration and invasion. The levels of MMP-2, MMP-9, E-cad and integrin β1 in the TPA-induced A549 cells changed markedly, compared with the untreated cells. In addition, the suppression of Cal was affected by the PKC inhibitor, AEB071, an ERK1/2 inhibitor, PD98059. The results of the present study indicated that Cal inhibited the proliferation, adhesion, migration and invasion of the TPA-induced A549 cells. The Cal-induced repression of PKC-α/ERK1/2, increased the expression of E-Cad and inhibited the expression levels of MMP-2, MMP-9 and integrin β1, which possibly

  7. Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts.

    PubMed

    Svegliati, Silvia; Cancello, Raffaella; Sambo, Paola; Luchetti, Michele; Paroncini, Paolo; Orlandini, Guido; Discepoli, Giancarlo; Paterno, Roberto; Santillo, Mariarosaria; Cuozzo, Concetta; Cassano, Silvana; Avvedimento, Enrico V; Gabrielli, Armando

    2005-10-28

    The levels of Ras proteins in human primary fibroblasts are regulated by PDGF (platelet-derived growth factor). PDGF induced post-transcriptionally Ha-Ras by stimulating reactive oxygen species (ROS) and ERK1/2. Activation of ERK1/2 and high ROS levels stabilize Ha-Ras protein, by inhibiting proteasomal degradation. We found a remarkable example in vivo of amplification of this circuitry in fibroblasts derived from systemic sclerosis (scleroderma) lesions, producing vast excess of ROS and undergoing rapid senescence. High ROS, Ha-Ras, and active ERK1/2 stimulated collagen synthesis, DNA damage, and accelerated senescence. Conversely ROS or Ras inhibition interrupted the signaling cascade and restored the normal phenotype. We conclude that in primary fibroblasts stabilization of Ras protein by ROS and ERK1/2 amplifies the response of the cells to growth factors and in systemic sclerosis represents a critical factor in the onset and progression of the disease. PMID:16081426

  8. Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

    PubMed

    Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

    2014-01-01

    We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb(P) CO17-1A × BR55-treated. The mAb(P) CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb(P) CO17-1A × BR55. In addition, the mAb(P) CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer. PMID:25405740

  9. Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

    PubMed

    Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

    2014-11-14

    We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb(P) CO17-1A × BR55-treated. The mAb(P) CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb(P) CO17-1A × BR55. In addition, the mAb(P) CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

  10. Growth Suppression of Colorectal Cancer by Plant-Derived Multiple mAb CO17-1A × BR55 via Inhibition of ERK1/2 Phosphorylation

    PubMed Central

    Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

    2014-01-01

    We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAbP) CO17-1A and mAbP CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAbP CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAbP CO17-1A × BR55-treated. The mAbP CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAbP CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAbP CO17-1A × BR55. In addition, the mAbP CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAbP CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAbP CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer. PMID:25405740

  11. Cinnamtannin D1 from Rhododendron formosanum Induces Autophagy via the Inhibition of Akt/mTOR and Activation of ERK1/2 in Non-Small-Cell Lung Carcinoma Cells.

    PubMed

    Way, Tzong-Der; Tsai, Shang-Jie; Wang, Chao-Min; Jhan, Yun-Lian; Ho, Chi-Tang; Chou, Chang-Hung

    2015-12-01

    In our previous study, ursolic acid present in the leaves of Rhododendron formosanum was found to possess antineoplastic activity. We further isolated and unveiled a natural product, cinnamtannin D1 (CNT D1), an A-type procyanidin trimer in R. formosanum also exhibiting anticancer efficacy that induced G1 arrest (83.26 ± 3.11% for 175 μM CNT D1 vs 69.28 ± 1.15% for control, p < 0.01) and autophagy in non-small-cell lung carcinoma (NSCLC) cells. We found that CNT D1-mediated autophagy was via the noncanonical pathway, being beclin-1-independent but Atg5 (autophagy-related genes 5)-dependent. Inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in CNT D1-treated NSCLC cells. Moreover, CNT D1 inhibited the Akt/mammalian target of the rapamycin (mTOR) pathway and activated the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, resulting in induction of autophagy.

  12. Up-regulated HMGB1 in EAM directly led to collagen deposition by a PKCβ/Erk1/2-dependent pathway: cardiac fibroblast/myofibroblast might be another source of HMGB1

    PubMed Central

    Su, Zhaoliang; Yin, Jingping; Wang, Ting; Sun, Yingkun; Ni, Ping; Ma, Rui; Zhu, Haitao; Zheng, Dong; Shen, Huiling; Xu, Wenlin; Xu, Huaxi

    2014-01-01

    High mobility group box 1 (HMGB1), an important inflammatory mediator, is actively secreted by immune cells and some non-immune cells or passively released by necrotic cells. HMGB1 has been implicated in many inflammatory diseases. Our previous published data demonstrated that HMGB1 was up-regulated in heart tissue or serum in experimental autoimmune myocarditis (EAM); HMGB1 blockade could ameliorate cardiac fibrosis at the last stage of EAM. And yet, until now, no data directly showed that HMGB1 was associated with cardiac fibrosis. Therefore, the aims of the present work were to assess whether (1) up-regulated HMGB1 could directly lead to cardiac fibrosis in EAM; (2) cardiac fibroblast/myofibroblasts could secrete HMGB1 as another source of high-level HMGB1 in EAM; and (3) HMGB1 blockade could effectively prevent cardiac fibrosis at the last stage of EAM. Our results clearly demonstrated that HMGB1 could directly lead to cardiac collagen deposition, which was associated with PKCβ/Erk1/2 signalling pathway; furthermore, cardiac fibroblast/myofibroblasts could actively secrete HMGB1 under external stress; and HMGB1 secreted by cardiac fibroblasts/myofibroblasts led to cardiac fibrosis via PKCβ activation by autocrine means; HMGB1 blockade could efficiently ameliorate cardiac fibrosis in EAM mice. PMID:24912759

  13. Exogenous hydrogen sulfide promotes C6 glioma cell growth through activation of the p38 MAPK/ERK1/2-COX-2 pathways.

    PubMed

    Zhen, Yulan; Zhang, Wei; Liu, Chujie; He, Jing; Lu, Yun; Guo, Ruixian; Feng, Jianqiang; Zhang, Ying; Chen, Jingfu

    2015-11-01

    Hydrogen sulfide (H2S) participates in multifarious physiological and pathophysiologic progresses of cancer both in vitro and in vivo. We have previously demonstrated that exogenous H2S promoted liver cancer cells proliferation/anti‑apoptosis/angiogenesis/migration effects via amplifying the activation of NF-κB pathway. However, the effects of H2S on cancer cell proliferation and apoptosis are controversial and remain unclear in C6 glioma cells. The present study investigated the effects of exogenous H2S on cancer cells growth via activating p38 MAPK/ERK1/2-COX-2 pathways in C6 glioma cells. C6 glioma cells were treated with 400 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated (p)-p38 MAPK, total (t)-p38 MAPK, p-ERK1/2, t-ERK1/2, cyclooxygenase-2 (COX-2) and caspase-3 were measured by western blotting assay. Cell viability was detected by Cell Counting Kit-8 (CCK-8). Apoptotic cells were observed by Hoechst 33258 staining assay. Cell proliferation was directly detected under fully automatic inverted microscope. Exposure of C6 glioma cells to NaHS resulted in cell proliferation, as evidenced by an increase in cell viability. In addition, NaHS treatment reduced apoptosis, as indicated by the decreased apoptotic percentage and the cleaved caspase-3 expression. Importantly, exposure of the cells to NaHS increased the expression levels of p-p38 MAPK, p-ERK1/2 and COX-2. Notably, co-treatment of C6 glioma cells with 400 µmol/l NaHS and AOAA (an inhibitor of CBS) largely suppressed the above NaHS-induced effects. Combined treatment with NaHS and SB203580 (an inhibitor of p38 MAPK) or PD-98059 (an inhibitor of ERK1/2) resulted in the synergistic reduction of COX-2 expression and increase of caspase-3 expression, a decreased number of apoptotic cells, along with decreased cell viability. Combined treatment with NS-398 (an inhibitor of COX-2) and NaHS also resulted in the synergistic increase of caspase-3, a decreased in the

  14. ERK1/2 signaling is essential for the chemoattraction exerted by human FGF2 and human anosmin-1 on newborn rat and mouse OPCs via FGFR1.

    PubMed

    Murcia-Belmonte, Verónica; Medina-Rodríguez, Eva M; Bribián, Ana; de Castro, Fernando; Esteban, Pedro F

    2014-03-01

    Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration, as well as differentiation events such as myelination. Anosmin-1 is an extracellular matrix (ECM) glycoprotein that interacts with the fibroblast growth factor receptor 1 (FGFR1) to exert its biological actions through this receptor, although the intracellular pathways underlying anosmin-1 signaling remain largely unknown. This protein is defective in the X-linked form of Kallmann syndrome (KS) and has a prominent role in the migration of neuronal and oligodendroglial precursors. We have shown that anosmin-1 exerts a chemotactic effect via FGFR1 on neuronal precursors from the subventricular zone (SVZ) and the essential role of the ERK1/2 signaling. We report here the positive chemotactic effect of FGF2 and anosmin-1 on rat and mouse postnatal OPCs via FGFR1. The same effect was observed with the truncated N-terminal region of anosmin-1 (A1Nt). The introduction in anosmin-1 of the missense mutation F517L found in patients suffering from KS annulled the chemotactic activity; however, the mutant form carrying the disease-causing mutation E514K also found in KS patients, behaved as the wild-type protein. The chemoattraction exhibited by FGF2 and anosmin-1 on OPCs was blocked by the mitogen-activated protein kinase (MAPK) inhibitor U0126, suggesting that the activation of the ERK1/2 MAPK signaling pathway following interaction with the FGFR1 is necessary for FGF2 and anosmin-1 to exert their chemotactic effect. In fact, both proteins were able to induce the phosphorylation of the ERK1/2 kinases after the activation of the FGFR1 receptor.

  15. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

    PubMed

    Soares, Ana Sofia; Costa, Vera Marisa; Diniz, Carmen; Fresco, Paula

    2015-01-01

    Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression.

  16. Tumor Necrosis Factor Alpha Inhibits L-Type Ca2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

    PubMed Central

    Reyes-García, Jorge; Flores-Soto, Edgar; Solís-Chagoyán, Héctor; Sommer, Bettina; Díaz-Hernández, Verónica; García-Hernández, Luz María

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca2+ channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway. PMID:27445440

  17. Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

    SciTech Connect

    Kim, Tack-Joong; Kang, Yeo-Jin; Lim, Yong; Lee, Hyoung-Woo; Bae, Kiho; Lee, Youn-Sun; Yoo, Jae-Myung; Yoo, Hwan-Soo; Yun, Yeo-Pyo

    2011-08-15

    Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [{sup 3}H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

  18. Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

    PubMed

    Reyes-García, Jorge; Flores-Soto, Edgar; Solís-Chagoyán, Héctor; Sommer, Bettina; Díaz-Hernández, Verónica; García-Hernández, Luz María; Montaño, Luis M

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

  19. Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

    PubMed

    Reyes-García, Jorge; Flores-Soto, Edgar; Solís-Chagoyán, Héctor; Sommer, Bettina; Díaz-Hernández, Verónica; García-Hernández, Luz María; Montaño, Luis M

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway. PMID:27445440

  20. Increasing progranulin levels and blockade of the ERK1/2 pathway: upstream and downstream strategies for the treatment of progranulin deficient frontotemporal dementia.

    PubMed

    Alquezar, Carolina; Esteras, Noemí; de la Encarnación, Ana; Moreno, Fermín; López de Munain, Adolfo; Martín-Requero, Ángeles

    2015-03-01

    Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disorder marked by mild-life onset and progressive changes in behavior, social cognition, and language. Loss-of-function progranulin gene (GRN) mutations are the major cause of FTLD with TDP-43 protein inclusions (FTLD-TDP). Disease-modifying treatments for FTLD-TDP are not available yet. Mounting evidence indicates that cell cycle dysfunction may play a pathogenic role in neurodegenerative disorders including FTLD. Since cell cycle re-entry of posmitotic neurons seems to precede neuronal death, it was hypothesized that strategies aimed at preventing cell cycle progression would have neuroprotective effects. Recent research in our laboratory revealed cell cycle alterations in lymphoblasts from FTLD-TDP patients carrying a null GRN mutation, and in PGRN deficient SH-SY5Y neuroblastoma cells, involving overactivation of the ERK1/2 signaling pathway. In this work, we have investigated the effects of PGRN enhancers drugs and ERK1/2 inhibitors, in these cellular models of PGRN-deficient FTLD. We report here that both restoring the PGRN content, by suberoylanilide hydroxamic acid (SAHA) or chloroquine (CQ), as blocking ERK1/2 activation by selumetinib (AZD6244) or MEK162 (ARRY-162), normalized the CDK6/pRb pathway and the proliferative activity of PGRN deficient cells. Moreover, we found that SAHA and selumetinib prevented the cytosolic TDP-43 accumulation in PGRN-deficient lymphoblasts. Considering that these drugs are able to cross the blood-brain barrier, and assuming that the alterations in cell cycle and signaling observed in lymphoblasts from FTLD patients could be peripheral signs of the disease, our results suggest that these treatments may serve as novel therapeutic drugs for FTLD associated to GRN mutations.

  1. Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways

    PubMed Central

    Nagayama, Kosuke; Kyotani, Yoji; Zhao, Jing; Ito, Satoyasu; Ozawa, Kentaro; Bolstad, Francesco A.; Yoshizumi, Masanori

    2015-01-01

    Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus. PMID:26379274

  2. PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts.

    PubMed

    Sun, Qingzhu; Liu, Li; Mandal, Jyotshna; Molino, Antonio; Stolz, Daiana; Tamm, Michael; Lu, Shemin; Roth, Michael

    2016-04-01

    Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases.

  3. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

    PubMed

    Soares, Ana Sofia; Costa, Vera Marisa; Diniz, Carmen; Fresco, Paula

    2015-01-01

    Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression. PMID:24909096

  4. Sirolimus and cyclosporine A alter barrier function in renal proximal tubular cells through stimulation of ERK1/2 signaling and claudin-1 expression.

    PubMed

    Martin-Martin, Natalia; Ryan, Gavin; McMorrow, Tara; Ryan, Michael P

    2010-03-01

    Alteration of the tight junction complex in renal epithelial cells can affect renal barrier function and perturb normal kidney homeostasis. The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation but this combination can also result in increased adverse effects. We previously showed that CsA treatment alone caused an alteration of the tight junction complex, resulting in changes in transepithelial permeability in Madin-Darby canine kidney distal tubular/collecting duct cells. The potential effect of SRL on transepithelial permeability in kidney cells is unknown. In this study, subcytotoxic doses of SRL or CsA were found to decrease the paracellular permeability of the porcine proximal tubular epithelial cells, LLC-PK1 cell monolayers, which was detected as an increase in transepithelial electrical resistance (TER). The cotreatment with SRL and CsA was found to increase TER in a synergistic manner. CsA treatment increased total cellular expression and membrane localization of the tight junction protein claudin-1 and this further increased with the combination of SRL/CsA. SRL and CsA treatment alone or in combination stimulated the phosphorylation of ERK1/2. The MEK-ERK1/2 pathway inhibitor, U0126, reduced the SRL, CsA, and CsA/SRL-induced increase in TER. U0126 also reduced the CsA and CsA/SRL-induced increase in the membrane localization of claudin-1. Alterations in claudin-2 and claudin-4 were also detected. However, the results suggest that the modulation in expression and localization of claudin-1 appears to be pivotal in the SRL- and CsA-induced modulation of the epithelial barrier function and that modulation is regulated by ERK1/2 signaling pathway. PMID:19955189

  5. 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. PMID:17898229

  6. 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.

  7. Erratum: Mannose-capped lipoarabinomannan from Mycobacterium tuberculosis induces IL-37 production via upregulating ERK1/2 and p38 in human type II alveolar epithelial cells.

    PubMed

    Huang, Zhen; Zhao, Gao Wei; Gao, Chun Hai; Chi, Xiu Wen; Zeng, Tao; Hu, Yan Wei; Zheng, Lei; Wang, Qian

    2015-01-01

    The major surface lipoglycan of Mycobacterium tuberculosis (M. tb), mannose-capped lipoarabinomannan (ManLAM), is an immunosuppressive epitope of M. tb. Interleukin (IL)-37, is a newly identified anti-inflammatory cytokine, which reduces systemic and local inflammation. However, the correlation between ManLAM and IL-37 remains unknown. Therefore, in this study, we investigate the possible role and relative molecular mechanism of ManLAM in IL-37 production of human type II alveolar epithelial cells by using A549 cell line. Here, we report that M. tb induced IL-37 mRNA and protein expression in a time-dependent manner. We next fractionated components of M. tb using chloroform: methanol (C:M) and water. In sharp contrast to the C:M phase, water phase was mainly responsible for the production of IL-37. Since ManLAM is the major component of water phase, we found that ManLAM induced IL-37 mRNA and protein expression in a time and dose-dependent manner, while this activity was almost totally abolished by the ERK1/2 (U0126) and p38 (SB203580) inhibitor. ManLAM stimulation significantly induced ERK1/2 and p38 phosphorylation in A549 cells, as well as cell surface TLR2 expression. After interfering TLR2 expression, ERK1/2 and p38 phosphorylation levels were markedly decreased, and also IL-37 production. Though ManLAM also promoted TLR4 expression on A549 cells, TLR4 interference showed no influence on ManLAM-induced IL-37 production. Our results indicate that ManLAM induces IL-37 production in human type II alveolar epithelial cells via up-regulating TLR2/p38 or ERK1/2 pathway, and this provide an important evidence to explain the pathological role of ManLAM that contribute to the persistence of M. tb.[This corrects the article on p. 7279 in vol. 8, PMID: 26221267.]. PMID:26770645

  8. 5,14-HEDGE, a 20-HETE mimetic, reverses hypotension and improves survival in a rodent model of septic shock: Contribution of soluble epoxide hydrolase, CYP2C23, MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway, and proinflammatory cytokine formation

    PubMed Central

    Tunctan, Bahar; Korkmaz, Belma; Sari, Ayse Nihal; Kacan, Meltem; Unsal, Demet; Serin, Mehmet Sami; Buharalioglu, C. Kemal; Sahan-Firat, Seyhan; Cuez, Tuba; Schunck, Wolf-Hagen; Falck, John R.; Malik, Kafait U.

    2013-01-01

    We have previously demonstrated that a stable synthetic analog of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), restores vascular reactivity, blood pressure, and heart rate in endotoxemic rats. The aim of this study was to determine whether decreased renal expression and activity of soluble epoxide hydrolase (sEH), MEK1, ERK1/2, IKKβ, IκB-α, and NF-κB as well as systemic and renal proinflammatory cytokine production associated with increased expression and activity of CYP2C23 contributes to the effect of 5,14-HEDGE to prevent hypotension, tachycardia, inflammation, and mortality in response to systemic administration of lipopolysaccharide (LPS). Blood pressure fell by 33 mmHg and heart rate rose by 57 beats/min in LPS (10 mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of sEH associated with a decrease in CYP2C23 mRNA and protein expression. Increased activity of sEH and p-MEK1, p-ERK1/2, p-IκB-α, NF-κB, and p-NF-κB protein levels as well as TNF-α and IL-8 production by LPS were also associated with a decreased activity of AA epoxygenases. These effects of LPS were prevented by 5,14-HEDGE (30 mg/kg, s.c.; 1 h after LPS). Treatment of endotoxemic mice with 5,14-HEDGE also raised the survival rate of animals from 84% to 98%. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, 20-HEDE (30 mg/kg, s.c.; 1 h after LPS) prevented the effects of 5,14-HEDGE on blood pressure, heart rate, expression and/or activity of sEH, CYP2C23, and ERK1/2 as well as TNF-α and IL-8 levels in rats treated with LPS. These results suggest that decreased expression and/or activity of sEH and MEK1/ERK1/2/IKKβ/IκB-α/NF-κB pathway as well as proinflammatory cytokine production associated with increased CYP2C23 expression and antiinflammatory mediator formation participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, inflammation, and

  9. OPN Induces FoxM1 Expression and Localization through ERK 1/2, AKT, and p38 Signaling Pathway in HEC-1A Cells

    PubMed Central

    Xie, Yunpeng; Li, Yinghua; Kong, Ying

    2014-01-01

    Mammalian embryo implantation is an extremely complex process and requires endometrial receptivity. In order to establish this receptivity, sequential proliferation and differentiation during the menstrual cycle is necessary. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion and progression. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. Osteopontin (OPN), an adhesion molecule, has been studied extensively in reproduction. In this study, we observed the expression and distribution of FoxM1 during the proliferative-phase and secretory-phase human endometrium and the pre-implantation mouse uterus firstly. Then we observed the relationship between OPN and FoxM1. Our results showed that FoxM1 was mainly distributed in glandular epithelium. OPN increased the expression of FoxM1 in the human uterine epithelial cell line HEC-1A cells in a time- and concentration-dependent manner. OPN regulates FoxM1 to influence HEC-1A cell proliferation through extracellular regulated protein kinases (ERK 1/2), protein kinase B (PKB, AKT), and the p38 mitogen activated protein kinases (p38MAPK, p38) signaling pathway. Inhibition of ERK 1/2, AKT and p38 suppressed OPN-induced FoxM1 expression and location. Our data indicate that FoxM1 might be regulated by OPN to influence endometrial proliferation to establish endometrial receptivity. PMID:25522167

  10. OPN induces FoxM1 expression and localization through ERK 1/2, AKT, and p38 signaling pathway in HEC-1A cells.

    PubMed

    Xie, Yunpeng; Li, Yinghua; Kong, Ying

    2014-12-16

    Mammalian embryo implantation is an extremely complex process and requires endometrial receptivity. In order to establish this receptivity, sequential proliferation and differentiation during the menstrual cycle is necessary. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion and progression. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. Osteopontin (OPN), an adhesion molecule, has been studied extensively in reproduction. In this study, we observed the expression and distribution of FoxM1 during the proliferative-phase and secretory-phase human endometrium and the pre-implantation mouse uterus firstly. Then we observed the relationship between OPN and FoxM1. Our results showed that FoxM1 was mainly distributed in glandular epithelium. OPN increased the expression of FoxM1 in the human uterine epithelial cell line HEC-1A cells in a time- and concentration-dependent manner. OPN regulates FoxM1 to influence HEC-1A cell proliferation through extracellular regulated protein kinases (ERK 1/2), protein kinase B (PKB, AKT), and the p38 mitogen activated protein kinases (p38MAPK, p38) signaling pathway. Inhibition of ERK 1/2, AKT and p38 suppressed OPN-induced FoxM1 expression and location. Our data indicate that FoxM1 might be regulated by OPN to influence endometrial proliferation to establish endometrial receptivity.

  11. Paeonol Inhibits Proliferation of Vascular Smooth Muscle Cells Stimulated by High Glucose via Ras-Raf-ERK1/2 Signaling Pathway in Coculture Model

    PubMed Central

    Chen, Junjun; Dai, Min; Wang, Yueqin

    2014-01-01

    Paeonol (Pae) has been previously reported to protect against atherosclerosis (AS) by inhibiting vascular smooth muscle cell (VSMC) proliferation or vascular endothelial cell (VEC) injury. But studies lack how VSMCs and VECs interact when Pae plays a role. The current study was based on a coculture model of VSMCs and VECs to investigate the protective mechanisms of Pae on atherosclerosis (AS) by determining the secretory function of VECs and proliferation of VSMCs focusing on the Ras-Raf-ERK1/2 signaling pathway. VECs were stimulated by high glucose. Our data showed that high concentration (35.5 mM) of glucose induced damage in VECs. Injury of VECs stimulated VSMC proliferation in the coculture model. Pae (120 μM) decreased vascular endothelial growth factor (VEGF) and platelet derivative growth factor B (PDGF-B) release from VECs and inhibited overexpression of Ras, P-Raf, and P-ERK proteins in VSMCs. The results indicate that diabetes modulates the inflammatory response in VECs to stimulate VSMC proliferation and promote the development of AS. Pae was beneficial by inhibiting the inflammatory effects of VECs on VSMC proliferation. This study suggests the inhibitory mechanism of Pae due to the inhibition of VEGF and PDGF-B secretion in VECs and Ras-Raf-ERK1/2 signaling pathway in VSMCs. PMID:25002903

  12. CXCL3 contributes to CD133+ CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

    PubMed Central

    Zhang, Lin; Zhang, Lixing; Li, Hong; Ge, Chao; Zhao, Fangyu; Tian, Hua; Chen, Taoyang; Jiang, Guoping; Xie, Haiyang; Cui, Ying; Yao, Ming; Li, Jinjun

    2016-01-01

    Although the chemotactic cytokine CXCL3 is thought to play an important role in tumor initiation and invasion, little is known about its function in hepatocellular carcinoma (HCC). In our previous study, we found that Ikaros inhibited CD133 expression via the MAPK pathway in HCC. Here, we showed that Ikaros may indirectly down-regulate CXCL3 expression in HCC cells, which leads to better outcomes in patients with CD133+ cancer stem cell (CSC) populations. CD133 overexpression induced CXCL3 expression, and silencing of CD133 down-regulated CXCL3 in HCC cells. Knockdown of CXCL3 inhibited CD133+ HCC CSCs’ self-renewal and tumorigenesis. The serum CXCL3 level was higher in HCC patients’ samples than that in healthy individual. HCC patients with higher CXCL3 expression displayed a poor prognosis, and a high level of CXCL3 was significantly associated with vascular invasion and tumor capsule formation. Exogenous CXCL3 induced Erk1/2 and ETS1 phosphorylation and promoted CD133 expression, indicating a positive feedback loop between CXCL3 and CD133 gene expression in HCC cells via Erk1/2 activation. Together, our findings indicated that CXCL3 might be a potent therapeutic target for HCC. PMID:27255419

  13. (+)-2-(1-Hydroxyl-4-oxocyclohexyl) ethyl caffeate suppresses solar UV-induced skin carcinogenesis by targeting PI3K, ERK1/2, and p38.

    PubMed

    Lim, Do Young; Lee, Mee-Hyun; Shin, Seung Ho; Chen, Hanyoung; Ryu, Joohyun; Shan, Lei; Li, Honglin; Bode, Ann M; Zhang, Wei-Dong; Dong, Zigang

    2014-08-01

    For decades, skin cancer incidence has increased, mainly because of oncogenic signaling pathways activated by solar ultraviolet (UV) irradiation (i.e., sun exposure). Solar UV induces multiple signaling pathways that are critical in the development of skin cancer, and therefore the development of compounds capable of targeting multiple molecules for chemoprevention of skin carcinogenesis is urgently needed. Herein, we examined the chemopreventive effects and the molecular mechanism of (+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate (HOEC), isolated from Incarvillea mairei var. grandiflora (Wehrhahn) Grierson. HOEC strongly inhibited neoplastic transformation of JB6 Cl41 cells without toxicity. PI3K, ERK1/2, and p38 kinase activities were suppressed by direct binding with HOEC in vitro. Our in silico docking data showed that HOEC binds at the ATP-binding site of each kinase. The inhibition of solar UV-induced PI3K, ERK1/2, and p38 kinase activities resulted in suppression of their downstream signaling pathways and AP1 and NF-κB transactivation in JB6 cells. Furthermore, topical application of HOEC reduced skin cancer incidence and tumor volume in SKH-1 hairless mice chronically exposed to solar UV. In summary, our results show that HOEC exerts inhibitory effects on multiple kinase targets and their downstream pathways activated by solar UV in vitro and in vivo. These findings suggest that HOEC is a potent chemopreventive compound against skin carcinogenesis caused by solar UV exposure.

  14. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    PubMed

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

  15. Allium cepa Extract and Quercetin Protect Neuronal Cells from Oxidative Stress via PKC-ε Inactivation/ERK1/2 Activation

    PubMed Central

    2016-01-01

    Oxidative stress plays an important role in the pathophysiology of various neurologic disorders. Allium cepa extract (ACE) and their main flavonoid component quercetin (QCT) possess antioxidant activities and protect neurons from oxidative stress. We investigated the underlying molecular mechanisms, particularly those linked to the antioxidant effects of the ACE. Primary cortical neuronal cells derived from mouse embryos were preincubated with ACE or QCT for 30 min and exposed to L-buthionine sulfoximine for 4~24 h. We found that ACE and QCT significantly decreased neuronal death and the ROS increase induced by L-buthionine-S, R-sulfoximine (BSO) in a concentration-dependent manner. Furthermore, ACE and QCT activated extracellular signal-regulated kinase 1/2 (ERK1/2), leading to downregulation of protein kinase C-ε (PKC-ε) in BSO-stimulated neuronal cells. In addition, ACE and QCT decreased the phosphorylated levels of p38 mitogen-activated protein kinase. Our results provide new insight into the protective mechanism of ACE and QCT against oxidative stress in neuronal cells. The results suggest that the inactivation of PKC-ε induced by phosphorylating ERK1/2 is responsible for the neuroprotective effect of ACE and QCT against BSO-induced oxidative stress. PMID:27668036

  16. GPR30 decreases with vascular aging and promotes vascular smooth muscle cells maintaining differentiated phenotype and suppressing migration via activation of ERK1/2

    PubMed Central

    Huang, Fang; Yin, Jianguo; Li, Keyu; Li, Ying; Qi, Heng; Fang, Li; Yuan, Cong; Liu, Weiwei; Wang, Min; Li, Xiangping

    2016-01-01

    Estrogen receptors, including classic nuclear receptors ERα, ERβ, and membrane receptor GPR30, are expressed in vascular tissues and exert protective actions in vascular diseases. But the expression pattern and functional roles of GPR30 in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we found that ERα, ERβ, and GPR30 were decreased with VSMCs passaging in vitro or growing in vivo and activation of GPR30 promoted ERα expression. Then, we validated that activation of GPR30 significantly decreased migratory capability of VSMCs and suppressed ERα, whereas PDGF-BB (20 ng/mL) treatment caused increase of migration. And activation of GPR30 led to reduction of osteopontin and cellular retinol binding protein 1, enhancement of calponin and 3F8, and upregulation of total and phosphorylated ERK1/2 expression in VSMCs knocked down by GPR30, ERα, and ERβ or treated with PDGF-BB. These data suggest that GPR30 promotes VSMCs reducing migration and maintaining differentiated phenotype via activation of ERK1/2 pathway. Our findings provide novel mechanisms of GPR30 protection of VSMCs as well as a new target for prevention of vascular aging. PMID:27354813

  17. Rhus coriaria induces senescence and autophagic cell death in breast cancer cells through a mechanism involving p38 and ERK1/2 activation

    PubMed Central

    El Hasasna, Hussain; Athamneh, Khawlah; Al Samri, Halima; Karuvantevida, Noushad; Al Dhaheri, Yusra; Hisaindee, Soleiman; Ramadan, Gaber; Al Tamimi, Nedaa; AbuQamar, Synan; Eid, Ali; Iratni, Rabah

    2015-01-01

    Here, we investigated the anticancer effect of Rhus coriaria on three breast cancer cell lines. We demonstrated that Rhus coriaria ethanolic extract (RCE) inhibits the proliferation of these cell lines in a time- and concentration-dependent manner. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, downregulation of cyclin D1, p27, PCNA, c-myc, phospho-RB and expression of senescence-associated β-galactosidase activity. No proliferative recovery was detected after RCE removal. Annexin V staining and PARP cleavage analysis revealed a minimal induction of apoptosis in MDA-MB-231 cells. Electron microscopy revealed the presence of autophagic vacuoles in RCE-treated cells. Interestingly, blocking autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death and senescence. RCE was also found to activate p38 and ERK1/2 signaling pathways which coincided with induction of autophagy. Furthermore, we found that while both autophagy inhibitors abolished p38 phosphorylation, only CQ led to significant decrease in pERK1/2. Finally, RCE induced DNA damage and reduced mutant p53, two events that preceded autophagy. Our findings provide strong evidence that R. coriaria possesses strong anti-breast cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against breast cancer. PMID:26263881

  18. OPN induces FoxM1 expression and localization through ERK 1/2, AKT, and p38 signaling pathway in HEC-1A cells.

    PubMed

    Xie, Yunpeng; Li, Yinghua; Kong, Ying

    2014-01-01

    Mammalian embryo implantation is an extremely complex process and requires endometrial receptivity. In order to establish this receptivity, sequential proliferation and differentiation during the menstrual cycle is necessary. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion and progression. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. Osteopontin (OPN), an adhesion molecule, has been studied extensively in reproduction. In this study, we observed the expression and distribution of FoxM1 during the proliferative-phase and secretory-phase human endometrium and the pre-implantation mouse uterus firstly. Then we observed the relationship between OPN and FoxM1. Our results showed that FoxM1 was mainly distributed in glandular epithelium. OPN increased the expression of FoxM1 in the human uterine epithelial cell line HEC-1A cells in a time- and concentration-dependent manner. OPN regulates FoxM1 to influence HEC-1A cell proliferation through extracellular regulated protein kinases (ERK 1/2), protein kinase B (PKB, AKT), and the p38 mitogen activated protein kinases (p38MAPK, p38) signaling pathway. Inhibition of ERK 1/2, AKT and p38 suppressed OPN-induced FoxM1 expression and location. Our data indicate that FoxM1 might be regulated by OPN to influence endometrial proliferation to establish endometrial receptivity. PMID:25522167

  19. Sprouty2 Suppresses Epithelial-Mesenchymal Transition of Human Lens Epithelial Cells through Blockade of Smad2 and ERK1/2 Pathways

    PubMed Central

    Chen, Chuan; Chen, Xiaoyun; Qin, Yingyan; Qu, Bo; Luo, Lixia; Lin, Haotian; Wu, Mingxing; Chen, Weirong; Liu, Yizhi

    2016-01-01

    Transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a key role in the pathogenesis of anterior subcapsular cataract (ASC) and capsule opacification. In mouse lens, Sprouty2 (Spry2) has a negative regulatory role on TGFβ signaling. However, the regulation of Spry2 during ASC development and how Spry2 modulates TGFβ signaling pathway in human LECs have not been characterized. Here, we demonstrate that Spry2 expression level is decreased in anterior capsule LECs of ASC patients. Spry2 negatively regulates TGFβ2-induced EMT and migration of LECs through inhibition of Smad2 and ERK1/2 phosphorylation. Also, blockade of Smad2 or ERK1/2 activation suppresses EMT caused by Spry2 downregulation. Collectively, our results for the first time show in human LECs that Spry2 has an inhibitory role in TGFβ signaling pathway. Our findings in human lens tissue and epithelial cells suggest that Spry2 may become a novel therapeutic target for the prevention and treatment of ASC and capsule opacification. PMID:27415760

  20. Diastereomeric mixture of calophyllic acid and isocalophyllic acid stimulates glucose uptake in skeletal muscle cells: involvement of PI-3-kinase- and ERK1/2-dependent pathways.

    PubMed

    Prasad, Janki; Maurya, Chandan Kumar; Pandey, Jyotsana; Jaiswal, Natasha; Madhur, Gaurav; Srivastava, Arvind Kumar; Narender, Tadigoppula; Tamrakar, Akhilesh Kumar

    2013-05-01

    The diastereomeric mixture of calophyllic acid and isocalophyllic acid (F015) isolated from the leaves of Calophyllum inophyllum was investigated for the metabolic effect on glucose transport in skeletal muscle cells. In L6 myotubes, F015 dose-dependently stimulated glucose uptake by increasing translocation of glucose transporter4 (GLUT4) to plasma membrane without affecting their gene expression. The effects on glucose uptake were additive to insulin. Inhibitors analyses revealed that F015-induced glucose uptake was dependent on the activation of phosphatidylinositol-3-kinase (PI-3-K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), while independent to the activation of 5'AMP-activated kinase (AMPK). F015 significantly increased the phosphorylation of AKT, AS160 and ERK1/2, account for the augmented glucose transport capacity in L6 myotubes. Furthermore, F015 improved glucose tolerance and enhanced insulin sensitivity in skeletal muscle of dexamethasone-induced insulin resistant mice. Our findings demonstrate that F015 activates glucose uptake in skeletal muscle cells through PI-3-K- and EKR1/2-dependent mechanisms and can be a potential lead for the management of diabetes and obesity.

  1. Allium cepa Extract and Quercetin Protect Neuronal Cells from Oxidative Stress via PKC-ε Inactivation/ERK1/2 Activation

    PubMed Central

    2016-01-01

    Oxidative stress plays an important role in the pathophysiology of various neurologic disorders. Allium cepa extract (ACE) and their main flavonoid component quercetin (QCT) possess antioxidant activities and protect neurons from oxidative stress. We investigated the underlying molecular mechanisms, particularly those linked to the antioxidant effects of the ACE. Primary cortical neuronal cells derived from mouse embryos were preincubated with ACE or QCT for 30 min and exposed to L-buthionine sulfoximine for 4~24 h. We found that ACE and QCT significantly decreased neuronal death and the ROS increase induced by L-buthionine-S, R-sulfoximine (BSO) in a concentration-dependent manner. Furthermore, ACE and QCT activated extracellular signal-regulated kinase 1/2 (ERK1/2), leading to downregulation of protein kinase C-ε (PKC-ε) in BSO-stimulated neuronal cells. In addition, ACE and QCT decreased the phosphorylated levels of p38 mitogen-activated protein kinase. Our results provide new insight into the protective mechanism of ACE and QCT against oxidative stress in neuronal cells. The results suggest that the inactivation of PKC-ε induced by phosphorylating ERK1/2 is responsible for the neuroprotective effect of ACE and QCT against BSO-induced oxidative stress.

  2. Schwann cell proliferation and differentiation that is induced by ferulic acid through MEK1/ERK1/2 signalling promotes peripheral nerve remyelination following crush injury in rats

    PubMed Central

    Zhu, Xiaoyan; Li, Kun; Guo, Xin; Wang, Jian; Xiang, Yang

    2016-01-01

    Schwann cell proliferation and differentiation is critical for the remyelination of injured peripheral nerves. Ferulic acid (FA) is a widely used antioxidant agent with neuroprotective properties. However, the potentially beneficial effects of FA on Schwann cells are unknown. Therefore, the present study was designed to examine the effects of FA on Schwann cell proliferation and differentiation. By using the cultured primary Schwann cells and proliferation assay, the results identified that FA was capable of increasing Schwann cell proliferation and expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) in vitro. It was also observed that the beneficial effect of FA treatment on Schwann cells was mainly dependent on the activation of MEK1/ERK1/2 signalling. Furthermore, FA was intraperitoneally administered to rats with sciatic nerve crush injury, and the results revealed an increase in Schwann cell proliferation and differentiation, while the MAG and MBP expression levels in sciatic nerves were markedly upregulated following FA administration. In conclusion, the current results demonstrate that Schwann cell proliferation and differentiation is induced by FA through MEK1/ERK1/2 signalling and that FA may accelerate injured peripheral nerve remyelination. PMID:27588110

  3. Implication of Akt, ERK1/2 and alternative p38MAPK signalling pathways in human colon cancer cell apoptosis induced by green tea EGCG.

    PubMed

    Cerezo-Guisado, María Isabel; Zur, Rafal; Lorenzo, María Jesús; Risco, Ana; Martín-Serrano, Miguel A; Alvarez-Barrientos, Alberto; Cuenda, Ana; Centeno, Francisco

    2015-10-01

    We investigated apoptosis induced by the green tea component the epigallocatechin-3-gallate (EGCG) and the pathways underlying its activity in a colon cancer cell line. A complete understanding of the mechanism(s) and molecules targeted by green tea polyphenols could be useful in developing novel therapeutic approaches for cancer treatment. EGCG, which is the major polyphenol in green tea, has cytotoxic effects and induced cell death in HT-29 cell death. In this study, we evaluated the effect EGCG on mitogen-activated protein kinase (MAPK) and Akt pathways. EGCG treatment increased phospho-ERK1/2, -JNK1/2 and -p38α, -p38γ and -p38δ, as well as phospho-Akt levels. Using a combination of kinase inhibitors, we found that EGCG-induced cell death is partially blocked by inhibiting Akt, ERK1/2 or alternative p38MAPK activity. Our data suggest that these kinase pathways are involved in the anti-cancer effects of EGCG and indicate potential use of this compound as chemotherapeutic agent for colon cancer treatment.

  4. Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells.

    PubMed

    Zhu, Liqian; Ding, Xiuyan; Zhu, Xiaofang; Meng, Songshu; Wang, Jianye; Zhou, Hong; Duan, Qiangde; Tao, Jie; Schifferli, Dieter M; Zhu, Guoqiang

    2011-04-14

    Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1) infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.

  5. SP6616 as a new Kv2.1 channel inhibitor efficiently promotes β-cell survival involving both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways

    PubMed Central

    Zhou, T T; Quan, L L; Chen, L P; Du, T; Sun, K X; Zhang, J C; Yu, L; Li, Y; Wan, P; Chen, L L; Jiang, B H; Hu, L H; Chen, J; Shen, X

    2016-01-01

    Kv2.1 as a voltage-gated potassium (Kv) channel subunit has a pivotal role in the regulation of glucose-stimulated insulin secretion (GSIS) and pancreatic β-cell apoptosis, and is believed to be a promising target for anti-diabetic drug discovery, although the mechanism underlying the Kv2.1-mediated β-cell apoptosis is obscure. Here, the small molecular compound, ethyl 5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2–a]pyrimidine-6-carboxylate (SP6616) was discovered to be a new Kv2.1 inhibitor. It was effective in both promoting GSIS and protecting β cells from apoptosis. Evaluation of SP6616 on either high-fat diet combined with streptozocin-induced type 2 diabetic mice or db/db mice further verified its efficacy in the amelioration of β-cell dysfunction and glucose homeostasis. SP6616 treatment efficiently increased serum insulin level, restored β-cell mass, decreased fasting blood glucose and glycated hemoglobin levels, and improved oral glucose tolerance. Mechanism study indicated that the promotion of SP6616 on β-cell survival was tightly linked to its regulation against both protein kinases C (PKC)/extracellular-regulated protein kinases 1/2 (Erk1/2) and calmodulin(CaM)/phosphatidylinositol 3-kinase(PI3K)/serine/threonine-specific protein kinase (Akt) signaling pathways. To our knowledge, this may be the first report on the underlying pathway responsible for the Kv2.1-mediated β-cell protection. In addition, our study has also highlighted the potential of SP6616 in the treatment of type 2 diabetes. PMID:27148689

  6. Activation of GPR30 inhibits the growth of prostate cancer cells through sustained activation of Erk1/2, c-jun/c-fos-dependent upregulation of p21, and induction of G(2) cell-cycle arrest.

    PubMed

    Chan, Q K Y; Lam, H-M; Ng, C-F; Lee, A Y Y; Chan, E S Y; Ng, H-K; Ho, S-M; Lau, K-M

    2010-09-01

    G-protein-coupled receptor-30 (GPR30) shows estrogen-binding affinity and mediates non-genomic signaling of estrogen to regulate cell growth. We here showed for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited the growth of androgen-dependent and androgen-independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1 induced cell-cycle arrest at the G(2) phase and reduced the expression of G(2)-checkpoint regulators (cyclin-A2, cyclin-B1, cdc25c, and cdc2) and phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With extensive use of siRNA-knockdown experiments and the MEK inhibitor PD98059 in this study, we dissected the mechanism underlying G-1-induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G(2) phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa.

  7. Naturally Acquired HMW1- and HMW2-Specific Serum Antibodies in Adults and Children Mediate Opsonophagocytic Killing of Nontypeable Haemophilus influenzae

    PubMed Central

    Winter, Linda E.

    2015-01-01

    The HMW1 and HMW2 proteins are highly immunogenic adhesins expressed by approximately 75% of nontypeable Haemophilus influenzae (NTHi) strains, and HMW1- and HMW2-specific antibodies can mediate opsonophagocytic killing of NTHi. In this study, we assessed the ability of HMW1- and HMW2-specific antibodies in sera from healthy adults and convalescent-phase sera from children with NTHi otitis media to mediate killing of homologous and heterologous NTHi. The serum samples were examined pre- and postadsorption on HMW1 and HMW2 affinity columns, and affinity-purified antibodies were assessed for ability to mediate killing of homologous and heterologous strains. Adult serum samples mediated the killing of six prototype NTHi strains at titers of <1:10 to 1:1,280. HMW1- and HMW2-adsorbed sera demonstrated unchanged to 8-fold decreased opsonophagocytic titers against the homologous strains. Each affinity-purified antibody preparation mediated the killing of the respective homologous strain at titers of <1:10 to 1:320 and of the five heterologous strains at titers of <1:10 to 1:320, with most preparations killing most heterologous strains to some degree. None of the acute-phase serum samples from children mediated killing, but each convalescent-phase serum sample mediated killing of the infecting strain at titers of 1:40 to 1:640. HMW1- and HMW2-adsorbed convalescent-phase serum samples demonstrated ≥4-fold decreases in titer. Three of four affinity-purified antibody preparations mediated killing of the infecting strain at titers of 1:20 to 1:320, but no killing of representative heterologous strains was observed. HMW1- and HMW2-specific antibodies capable of mediating opsonophagocytic killing are present in the serum from normal adults and develop in convalescent-phase sera of children with NTHi otitis media. Continued investigation of the HMW1 and HMW2 proteins as potential vaccine candidates for the prevention of NTHi disease is warranted. PMID:26512048

  8. Relative Contribution of Dengue IgG Antibodies Acquired during Gestation or Breastfeeding in Mediating Dengue Disease Enhancement and Protection in Type I Interferon Receptor-Deficient Mice

    PubMed Central

    Lee, Pei Xuan; Ong, Li Ching; Libau, Eshele Anak; Alonso, Sylvie

    2016-01-01

    Dengue virus (DENV) causes a spectrum of diseases ranging from self-limiting dengue fever to severe conditions such as haemorrhagic fever and dengue shock syndrome. Antibody-dependent enhancement (ADE) is thought to explain the occurrence of severe dengue whereby pre-existing binding but non-neutralising antibodies enhance DENV infection. The ADE phenomenon is supported by epidemiological findings that infants that born to dengue immune mothers are at greater risk to develop severe dengue upon primary infection. The role of maternally acquired dengue-specific antibodies in disease enhancement was recently recapitulated in a mouse model where mice born to DENV1-immune mothers experienced enhanced disease severity upon DENV2 infection. Here, this study investigates the relative contribution of maternal dengue-specific antibodies acquired during gestation and breastfeeding in dengue disease. Using a surrogate breastfeeding mother experimental approach, we showed that majority of the maternal dengue-specific antibodies were acquired during breastfeeding and conferred an extended enhancement window. On the other hand, in the context of homologous infection, breastfeeding conferred protection. Furthermore, measurement of dengue-specific antibody titres over time in mice born to dengue immune mothers revealed a biphasic pattern of antibody decay as reported in humans. Our work provides evidence of the potential contribution of breast milk-acquired dengue-specific IgG antibodies in enhancement and protection against dengue. Should such contribution be established in humans as well, it may have important implications for the development of guidelines to dengue-immune breastfeeding mothers. PMID:27341339

  9. Relative Contribution of Dengue IgG Antibodies Acquired during Gestation or Breastfeeding in Mediating Dengue Disease Enhancement and Protection in Type I Interferon Receptor-Deficient Mice.

    PubMed

    Lee, Pei Xuan; Ong, Li Ching; Libau, Eshele Anak; Alonso, Sylvie

    2016-06-01

    Dengue virus (DENV) causes a spectrum of diseases ranging from self-limiting dengue fever to severe conditions such as haemorrhagic fever and dengue shock syndrome. Antibody-dependent enhancement (ADE) is thought to explain the occurrence of severe dengue whereby pre-existing binding but non-neutralising antibodies enhance DENV infection. The ADE phenomenon is supported by epidemiological findings that infants that born to dengue immune mothers are at greater risk to develop severe dengue upon primary infection. The role of maternally acquired dengue-specific antibodies in disease enhancement was recently recapitulated in a mouse model where mice born to DENV1-immune mothers experienced enhanced disease severity upon DENV2 infection. Here, this study investigates the relative contribution of maternal dengue-specific antibodies acquired during gestation and breastfeeding in dengue disease. Using a surrogate breastfeeding mother experimental approach, we showed that majority of the maternal dengue-specific antibodies were acquired during breastfeeding and conferred an extended enhancement window. On the other hand, in the context of homologous infection, breastfeeding conferred protection. Furthermore, measurement of dengue-specific antibody titres over time in mice born to dengue immune mothers revealed a biphasic pattern of antibody decay as reported in humans. Our work provides evidence of the potential contribution of breast milk-acquired dengue-specific IgG antibodies in enhancement and protection against dengue. Should such contribution be established in humans as well, it may have important implications for the development of guidelines to dengue-immune breastfeeding mothers. PMID:27341339

  10. LIV-1 suppression inhibits HeLa cell invasion by targeting ERK1/2-Snail/Slug pathway

    SciTech Connect

    Zhao Le; Chen Wei; Taylor, Kathryn M.; Cai Bin; Li Xu

    2007-11-09

    It was reported that expression of the estrogen-regulated zinc transporter LIV-1 was particularly high in human cervical cancer cell line HeLa. This result prompted us to study the role that LIV-1 played in human cervical cancer. The results of real-time PCR showed that LIV-1 mRNA was significantly higher in cervical cancer in situ than in normal tissues. RNAi mediated suppression of LIV-1 in HeLa cells significantly inhibited cell proliferation, colony formation, migration, and invasive ability, but had no effect on cell apoptosis. Furthermore, LIV-1 suppression is accompanied by down-regulation of p44/42 MAPK, phospho-p44/42 MAPK, Snail and Slug expression levels. Hence, our data provide the first evidence that LIV-1 mRNA is overexpressed in cervical cancer in situ and is involved in invasion of cervical cancer cells through targeting MAPK-mediated Snail and Slug expression.

  11. FADD adaptor and PEA-15/ERK1/2 partners in major depression and schizophrenia postmortem brains: basal contents and effects of psychotropic treatments.

    PubMed

    García-Fuster, M J; Díez-Alarcia, R; Ferrer-Alcón, M; La Harpe, R; Meana, J J; García-Sevilla, J A

    2014-09-26

    Enhanced brain apoptosis (neurons and glia) may be involved in major depression (MD) and schizophrenia (SZ), mainly through the activation of the intrinsic (mitochondrial) apoptotic pathway. In the extrinsic death pathway, pro-apoptotic Fas-associated death domain (FADD) adaptor and its non-apoptotic p-Ser194 FADD form have critical roles interacting with other death regulators such as phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) and extracellular signal-regulated kinase (ERK). The basal status of FADD (protein and messenger RNA (mRNA)) and the effects of psychotropic drugs (detected in blood/urine samples) were first assessed in postmortem prefrontal cortex of MD and SZ subjects (including a non-MD/SZ suicide group). In MD, p-FADD, but not total FADD (and mRNA), was increased (26%, n=24; all MD subjects) as well as p-FADD/FADD ratio (a pro-survival marker) in antidepressant-free MD subjects (50%, n=10). In contrast, cortical FADD (and mRNA), p-FADD, and p-FADD/FADD were not altered in SZ brains (n=21) regardless of antipsychotic medications (except enhanced mRNA in treated subjects). Similar negative results were quantified in the non-MD/SZ suicide group. In MD, the regulation of multifunctional PEA-15 (i.e., p-Ser116 PEA-15 blocks pro-apoptotic FADD and PEA-15 prevents pro-survival ERK action) and the modulation of p-ERK1/2 were also investigated. Cortical p-PEA-15 was not changed whereas PEA-15 was increased mainly in antidepressant-treated subjects (16-20%). Interestingly, cortical p-ERK<