Science.gov

Sample records for erk1 mediates acquired

  1. Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

    PubMed Central

    2013-01-01

    Background In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. Results To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2. Conclusions These findings

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

    PubMed Central

    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

    2015-01-01

    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. PMID:25761881

  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. 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. PMID:26616889

  5. 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. PMID:26542799

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

    PubMed Central

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

    2016-01-01

    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. PMID:26542799

  7. Hesperidin, A Popular Antioxidant Inhibits Melanogenesis via Erk1/2 Mediated MITF Degradation

    PubMed Central

    Lee, Heun Joo; Lee, Woo Jin; Chang, Sung Eun; Lee, Ga-Young

    2015-01-01

    Regulation of melanogenesis has been the focus of treatment for hyperpigmentary skin disorders. Although hesperidin is one of the most well-known, naturally occurring flavonoids with antioxidant and anti-inflammatory effect, its anti-melanogenic effect is not known. The present study aims to determine the anti-melanogenic effect of hespiridin as well as its underlying molecular mechanisms. Melanin contents were measured in normal human melanocytes and B16F10 melanoma cells. Protein and mRNA levels of tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1) and TRP-2 were determined. Melanogenesis-regulating signals were examined. In results, hesperidin strongly inhibited melanin synthesis and tyrosinase activity. Hesperidin decreased tyrosinase, TRP-1, and TRP-2 protein expression but increased phospho-extracellular signal-regulated kinase 1/2 (p-Erk1/2) expression. Specific inhibitor of Erk1/2 or proteasome inhibitor reversed the inhibition of melanogenesis induced by hesperidin. Taken together, hesperidin, a popular antioxidant, stimulated Erk1/2 phosphorylation which subsequently degraded MITF which resulted in suppression of melanogenic enzymes and melanin synthesis. PMID:26262610

  8. β-Adrenergic Receptor-Mediated Transactivation Of Epidermal Growth Factor Receptor Decreases Cardiomyocyte Apoptosis Through Differential Subcellular Activation of ERK1/2 and Akt

    PubMed Central

    Grisanti, Laurel A.; Talarico, Jennifer A.; Carter, Rhonda L.; Yu, Justine E.; Repas, Ashley A.; Radcliffe, Scott W.; Tang, Hoang-ai; Makarewich, Catherine A.; Houser, Steven R.; Tilley, Douglas G.

    2014-01-01

    Rationale β-adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to relay pro-survival effects via unknown mechanisms. Objective We hypothesized that acute βAR-mediated EGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promoting cell survival through modulation of apoptosis. Methods and Results C57BL/6 mice underwent acute i.p. injection with isoproterenol (ISO) ± AG 1478 (EGFR antagonist) to assess the impact of βAR-mediated EGFR transactivation on phosphorylation of ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 and P-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereas the P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in the plasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). βAR-mediated EGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL as well as caspase 3 activity/cleavage, which were sensitive to inhibition of either ERK1/2 (PD184352) or Akt (LY-294002) signaling. Caspase 3 activity/cleavage was also sensitive to inhibition of transcription, which, with an increase in nuclear P-ERK1/2 and P-Akt in response to ISO, suggested that βAR-mediated EGFR transactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) to be altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of both membrane-bound and soluble cardiomyocyte TRAIL levels. Conclusions βAR-mediated EGFR transactivation induces differential subcellular activation of ERK1/2 and Akt leading to increased cell survival through the modulation of caspase 3 activity and apoptotic gene expression in cardiomyocytes. PMID:24566221

  9. Tamoxifen Inhibits TGF-β-Mediated Activation of Myofibroblasts by Blocking Non-Smad Signaling Through ERK1/2.

    PubMed

    Carthy, Jon M; Sundqvist, Anders; Heldin, Angelos; van Dam, Hans; Kletsas, Dimitris; Heldin, Carl-Henrik; Moustakas, Aristidis

    2015-12-01

    Transforming growth factor-β (TGF-β) is a multifunctional cytokine which stimulates the differentiation of fibroblasts into myofibroblasts. Myofibroblasts are critical for normal wound healing, but also accumulate pathologically in a number of chronic inflammatory conditions where they are key contributors to aberrant tissue remodeling and fibrosis, and in cancer stroma. In the current study, we identified a role for tamoxifen as a potent inhibitor of the TGF-β-mediated activation of primary human skin and breast fibroblasts. Our data indicate that tamoxifen does not interfere with canonical Smad signaling downstream of TGF-β but rather blocks non-Smad signaling through ERK1/2 MAP-kinase and the AP-1 transcription factor FRA2. We further demonstrate by siRNA-mediated knockdown that FRA2 is critical for the induced expression of myogenic proteins in response to TGF-β. Functionally, TGF-β-stimulated fibroblast-mediated contraction of collagen gels was impaired in the presence of tamoxifen. Altogether, these data demonstrate that tamoxifen prevents myofibroblast differentiation and, therefore, may provide therapeutic benefits to patients suffering from chronic inflammatory conditions or cancer. PMID:26096876

  10. Extracellular Ca2+ Promotes Odontoblastic Differentiation of Dental Pulp Stem Cells via BMP2-Mediated Smad1/5/8 and Erk1/2 Pathways.

    PubMed

    Li, Shiting; Hu, Jing; Zhang, Gang; Qi, Wei; Zhang, Ping; Li, Pengfei; Zeng, Yong; Zhao, Wenfeng; Tan, Yinghui

    2015-09-01

    Ca(2+) is the main element of many pulp capping materials that are used to promote the regeneration of tertiary dentin, but the underlying molecular mechanism is not clear. In this study, we found that Ca(2+) increased the expression of the odontoblastic differentiation marker gene DSPP and promoted odontoblastic differentiation and mineralization of DPSCs, but inhibited ALP activity. Ca(2+) increases the expression of endogenous BMP2, which activates the Smad1/5/8 pathway and promotes the Smad1-Runx2 and Runx2-DSPP interaction in DPSCs. Inhibition of Smad1/5/8 with dorsomorphin partially blocked Runx2 activity; however, inhibition of the BMP2 receptor with Noggin nearly fully suppressed Runx2 activity. These results indicate that Ca(2+) promotes cell differentiation mainly via BMP2-mediated Smad-dependent and Smad-independent pathways. We then determined that the phosphorylation level of Erk1/2, but not JNK or p38, was significantly increased as a result of Ca(2+) stimulation. Blockage of Erk1/2 was found to inhibit Runx2 activity, indicating that Ca(2+) triggers the Erk1/2 pathway, which subsequently regulates Runx2 activity. In addition, inhibition of Erk1/2 differentially attenuated the phosphorylation levels of Smad1/5/8 and Smad2/3. Collectively, this study demonstrates that Ca(2+) activates the BMP2-mediated Smad1/5/8 and Erk1/2 pathways in DPSCs and that Smad1/5/8 and Erk1/2 signaling converge at Runx2 to control the odontoblastic differentiation of DPSCs. PMID:25656933

  11. Notoginsenoside R1-mediated neuroprotection involves estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways: a novel mechanism of Nrf2/ARE signaling activation.

    PubMed

    Meng, X; Sun, G; Ye, J; Xu, H; Wang, H; Sun, X

    2014-04-01

    Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has antioxidant and anti-apoptotic properties. Oxidative stress plays a pivotal role in neurodegenerative diseases. To mimic oxidative stress in neurons and explore the neuroprotection of NGR1, H₂O₂-induced neurotoxicity in NGF-induced differentiation of PC12 cells was used. In this study, NGR1 preconditioning provided neuroprotective effects via suppressing H₂O₂-induced the intracellular ROS accumulation, the increase in the product of lipid peroxidation (MDA), protein oxidation (protein carbonyl), and DNA fragmentation (8-OHdG), and mitochondrial membrane depolarization as well as caspase-3 activation. Moreover, NGR1 treatment alone potently increased the nuclear translocation of Nrf2, augmented ARE enhancer activity, and upregulated the expression and activity of phase II antioxidant enzymes including HO-1, NQO-1, and γ-GCSc. NGR1 could also increase the ERE activity and activate Akt and ERK1/2 pathways. NGR1-mediated activation of Nrf2/ARE signaling and neuroprotection were abolished by genetic silencing of Nrf2 using siRNA or the pharmacological blockade of estrogen receptors using ICI-182780, and partially inhibited by Akt siRNA or ERK siRNA transfection. In addition, the phosphorylation of ERK1/2 mediated by NGR1 was markedly inhibited in PC12 cells transfected with Akt siRNA. On the contrary, ERK1/2 siRNA transfection hardly had any effect on the phosphorylation of Akt mediated by NGR1. NGR1-mediated activation of Akt and ERK1/2 pathways was blocked by ICI-182780. In conclusion, NGR1 provided neuroprotection via inducing an estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways, subsequently activating Nrf2/ARE signaling and thereby up-regulating phase II antioxidant enzymes. PMID:24437944

  12. The effect of estrogen on the proliferation of endometrial cancer cells is mediated by ERRγ through AKT and ERK1/2.

    PubMed

    Sun, Yan; Wang, Cuicui; Yang, Hui; Ma, Xiaoxin

    2014-09-01

    The objective of this study was to explore the effects and underlying mechanism of estrogen-related receptor γ (ERRγ) on the proliferation of endometrial carcinoma cells. Ishikawa cells, human endometrial cancer cells, were treated with estrogen. Immunofluorescence was used to observe the expression of ERRγ. Stable transfection with the plasmid containing ERRγ shRNA was carried out to knock down the expression of ERRγ in Ishikawa cells. MTT assays were performed to analyze the proliferation of Ishikawa cells. The activation of extracellular signal-regulated protein kinase (ERK)1/2 and activated protein kinase B (AKT) signaling pathways was identified using specific phosphorylated antibodies against ERK1/2 and AKT. PD98059, a MEK inhibitor, and LY294002, a PI3K inhibitor, were used in the inhibition experiments. ERRγ could translocate from the cytoplasm to the nucleus in Ishikawa cells after exposure to estrogen. Knockdown of ERRγ inhibited estrogen-induced proliferation of Ishikawa cells. More interestingly, knockdown of ERRγ abolished the activation of ERK1/2 and AKT in the Ishikawa cells treated with estrogen. Inhibition of AKT in Ishikawa cells with LY294002 resulted in a significant reduction in the levels of phospho-ERK1/2, whereas inhibition of ERK1/2 with PD98059 exerted no effects on AKT activation. Our data showed that ERRγ mediated the effects of estrogen on the proliferation of endometrial cancer cells through the activation AKT and ERK1/2 signaling pathways, which indicated that ERRγ plays a key role in endometrial cancer. PMID:25068805

  13. 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. PMID:25876941

  14. ERK1/2-Egr-1 Signaling Pathway-Mediated Protective Effects of Electroacupuncture in a Mouse Model of Myocardial Ischemia-Reperfusion.

    PubMed

    Zhang, Juan; Song, Jiangang; Xu, Jin; Chen, Xuemei; Yin, Peihao; Lv, Xin; Wang, Xiangrui

    2014-01-01

    Early growth response- (Egr-) 1 is an upstream master switch in controlling inflammatory responses following myocardial ischemia-reperfusion (I/R). Activation of extracellular signal-regulated protein kinase-1 and kinase-2 (ERK1/2) signaling is known to upregulate Egr-1. ERK1/2 pathway has been previously shown to mediate the therapeutic action of electroacupucture (EA). Thus, we hypothesized that EA would reduce myocardial I/R injury and inflammatory responses through inhibiting Egr-1 expression via the ERK1/2 pathway. Mice were pretreated with EA, U0126, or combination of EA and U0126 and then underwent 1 h myocardial ischemia and 3 h reperfusion. We investigated that EA significantly attenuated the I/R-induced upregulation of both Egr-1 and phosporylated-ERK1/2 (p-ERK1/2), decreased myocardial inflammatory cytokines including tumor necrosis factor- α (TNF- α ) and interleukin-1 β (IL-1 β ), and reduced the infarct size and the release of cardiac troponin I (cTnI). U0126 treatment also exhibited the same effect as EA on Egr-1 level and subsequent cardioprotective effects. There was no additive effect of cotreatment with EA and U0126 on the expression of Egr-1 and its downstream target genes (TNF- α , IL-1 β ) or serum cTnI level. Collectively, these observations suggested that EA attenuates myocardial I/R injury, possibly through inhibiting the ERK1/2-Egr-1 signaling pathway and reducing the release of proinflammatory cytokines. PMID:24883066

  15. ERK1/2-Egr-1 Signaling Pathway-Mediated Protective Effects of Electroacupuncture in a Mouse Model of Myocardial Ischemia-Reperfusion

    PubMed Central

    Song, Jiangang; Xu, Jin; Chen, Xuemei; Lv, Xin

    2014-01-01

    Early growth response- (Egr-) 1 is an upstream master switch in controlling inflammatory responses following myocardial ischemia-reperfusion (I/R). Activation of extracellular signal-regulated protein kinase-1 and kinase-2 (ERK1/2) signaling is known to upregulate Egr-1. ERK1/2 pathway has been previously shown to mediate the therapeutic action of electroacupucture (EA). Thus, we hypothesized that EA would reduce myocardial I/R injury and inflammatory responses through inhibiting Egr-1 expression via the ERK1/2 pathway. Mice were pretreated with EA, U0126, or combination of EA and U0126 and then underwent 1 h myocardial ischemia and 3 h reperfusion. We investigated that EA significantly attenuated the I/R-induced upregulation of both Egr-1 and phosporylated-ERK1/2 (p-ERK1/2), decreased myocardial inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and reduced the infarct size and the release of cardiac troponin I (cTnI). U0126 treatment also exhibited the same effect as EA on Egr-1 level and subsequent cardioprotective effects. There was no additive effect of cotreatment with EA and U0126 on the expression of Egr-1 and its downstream target genes (TNF-α, IL-1β) or serum cTnI level. Collectively, these observations suggested that EA attenuates myocardial I/R injury, possibly through inhibiting the ERK1/2-Egr-1 signaling pathway and reducing the release of proinflammatory cytokines. PMID:24883066

  16. 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

  17. 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.

  18. MiR-126 promotes coxsackievirus replication by mediating cross-talk of ERK1/2 and Wnt/β-catenin signal pathways.

    PubMed

    Ye, Xin; Hemida, Maged Gomaa; Qiu, Ye; Hanson, Paul J; Zhang, Huifang Mary; Yang, Decheng

    2013-12-01

    Coxsackievirus B3 (CVB3) is one of the most prevalent causes of viral myocarditis and is associated with many other pathological conditions. CVB3 replication relies on host cellular machineries and causes direct damage to host cells. MicroRNAs have been found to regulate viral infections but their roles in CVB3 infection are still poorly understood. Here we describe a novel mechanism by which miR-126 regulates two signal pathways essential for CVB3 replication. We found that CVB3-induced ERK1/2 activation triggered the phosphorylation of ETS-1 and ETS-2 transcription factors, which induced miR-126 upregulation. By using both microRNA mimics and inhibitors, we proved that the upregulated miR-126 suppressed sprouty-related, EVH1 domain containing 1 (SPRED1) and in turn enhanced ERK1/2 activation. This positive feedback loop of ERK1/2-miR-126-ERK1/2 promoted CVB3 replication. Meanwhile, miR-126 expression stimulated GSK-3β activity and induced degradation of β-catenin through suppressing LRP6 and WRCH1, two newly identified targets in the Wnt/β-catenin pathway, which sensitized the cells to virus-induced cell death and increased viral progeny release to initiate new infections. Our results demonstrate that upregulated miR-126 upon CVB3 infection targets SPRED1, LRP6, and WRCH1 genes, mediating cross-talk between ERK1/2 and Wnt/β-catenin pathways, and thus promoting viral replication and contributes to the viral cytopathogenicity. PMID:23811937

  19. Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization.

    PubMed

    Wrzal, Paulina K; Devost, Dominic; Pétrin, Darlaine; Goupil, Eugénie; Iorio-Morin, Christian; Laporte, Stéphane A; Zingg, Hans H; Hébert, Terence E

    2012-01-01

    The oxytocin receptor (OTR) and the β(2)-adrenergic receptor (β(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects. PMID:21963428

  20. Acute effect of cigarette smoke on TNF-alpha release by macrophages mediated through the erk1/2 pathway.

    PubMed

    Demirjian, Loutfig; Abboud, Raja T; Li, Hong; Duronio, Vincent

    2006-06-01

    Pulmonary emphysema is a major cause of mortality and morbidity in chronic obstructive pulmonary disease (COPD). Cigarette smoking is a major risk factor in the development of pulmonary emphysema. In this study, we investigated the acute effect of cigarette smoke in vitro on the production of tumour necrosis factor-alpha (TNF-alpha) using differentiated U937 cells, a macrophage model system. We found that stimulation of the macrophages with cigarette smoke media (CSM) leads to a rapid activation of extracellular-regulated kinases 1 and 2 (erk1/2), p90RSK and a transient decrease in phosphorylation of PKB/akt. The CSM also caused the subsequent induction of TNF-alpha release. Our studies revealed that U0126, an inhibitor of the erk1/2 pathway, markedly suppressed CSM-induced TNF-alpha release. Consistent with this finding, U0126 blocked CSM-stimulated erk1/2 phosphorylation, as well as phosphorylation of the downstream kinase, p90RSK. On the other hand, the PI3-K inhibitor, LY294002, and epidermal growth factor receptor (EGFR)-specific inhibitor, AG1478, did not suppress the release of TNF-alpha. Thus, CSM induction of TNF-alpha production by differentiated macrophages is regulated primarily via the erk1/2 pathway. PMID:16777389

  1. 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

  2. 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.

  3. 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

  4. 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. PMID:27154044

  5. 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

  6. 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

  7. 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

  8. 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

  9. C23 protein meditates bone morphogenetic protein-2-mediated EMT via up-regulation of Erk1/2 and Akt in gastric cancer.

    PubMed

    Yang, Yonggang; Yang, Chunyan; Zhang, Jianping

    2015-03-01

    In our previous study, the epithelial-to-mesenchymal transition (EMT) has been identified to be involved in gastric cancer progression. Notably, nuclear protein C23 and bone morphogenetic protein-2 (BMP2) have been linked into EMT. However, the specific mechanisms underlying BMP2 pathway-mediated EMT are not still unraveled. In this study, we adopted immunohistochemistry and immunoblotting to determine the expression of C23 and BMP2 receptor II (BMPR-II) in 90 gastric cancer samples and cell lines. Subsequently, relevant cell lines were selected to be treated with si-C23 or si-BMPRII and the detection of in vitro assay. Our results revealed that both C23 and BMPRII were aberrantly and constitutively expressed in gastric cancer specimens and cell lines, whose expression was positively associated with metastasis, stage and differentiation, and portended poor survival outcome of gastric cancer patients. In vitro assay validated the increased expression of p-Erk1/2, p-Akt, vimentin, N-cadherin, and MMP2 in BMP2-stimulated MGC803 cells, which was in a dose-dependent manner. By contrast, si-C23 treatment attenuated the BMP2-stimulated expression of p-Erk1/2, p-Akt, vimentin, N-cadherin, and MMP2. Also, the treatment of either si-C23 or si-BMPRII decreased the ability of migration and invasion of MGC803 cells. In conclusion, C23 mediates BMP2-induced EMT progression via the up-regulation of Erk1/2 and Akt signaling pathway in gastric cancer, which indicated both C23 and BMPRII pathway could be recommended as prospective targets or biomarkers to antagonize the progression of gastric cancer. PMID:25698539

  10. Bisphenol AF-induced endogenous transcription is mediated by ERα and ERK1/2 activation in human breast cancer cells.

    PubMed

    Li, Ming; Guo, Jing; Gao, Wenhui; Yu, Jianlong; Han, Xiaoyu; Zhang, Jing; Shao, Bing

    2014-01-01

    Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ERα and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ERα-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ERα-positive T47D and MCF7 cells as well as overexpression of ERα in ERα-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ERα and ERK1/2 activation in human breast cancer cells. PMID:24727858

  11. Bisphenol AF-Induced Endogenous Transcription Is Mediated by ERα and ERK1/2 Activation in Human Breast Cancer Cells

    PubMed Central

    Li, Ming; Guo, Jing; Gao, Wenhui; Yu, Jianlong; Han, Xiaoyu; Zhang, Jing; Shao, Bing

    2014-01-01

    Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ERα and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ERα-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ERα-positive T47D and MCF7 cells as well as overexpression of ERα in ERα-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ERα and ERK1/2 activation in human breast cancer cells. PMID:24727858

  12. 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

  13. 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. PMID:26393302

  14. 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.

  15. 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

  16. Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells*

    PubMed Central

    Patel, Satyananda; Djerdjouri, Bahia; Raoul-Des-Essarts, Yannick; Dang, Pham My-Chan; El-Benna, Jamel; Périanin, Axel

    2010-01-01

    Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (Km 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67phox and p47phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47phox, p67phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase. PMID:20693286

  17. 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

  18. AHCYL1 is mediated by estrogen-induced ERK1/2 MAPK cell signaling and microRNA regulation to effect functional aspects of the avian oviduct.

    PubMed

    Jeong, Wooyoung; Kim, Jinyoung; Ahn, Suzie E; Lee, Sang In; Bazer, Fuller W; Han, Jae Yong; Song, Gwonhwa

    2012-01-01

    S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP(3) receptor-binding protein released with IP(3) (IRBIT), regulates IP(3)-induced Ca(2+) release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3'-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway. PMID:23145124

  19. Actin and ERK1/2-CEBPβ signaling mediates phagocytosis-induced innate immune response of osteoprogenitor cells.

    PubMed

    Lee, Heon Goo; Minematsu, Hiroshi; Kim, Kyung Ok; Celil Aydemir, Ayse B; Shin, Mike J; Nizami, Saqib A; Chung, Kook Jin; Hsu, Anny C; Jacobs, Christopher R; Lee, Francis Youngin

    2011-12-01

    Wear particles at the host bone-implant interface are a major challenge for successful bone implant arthoplasties. Current understanding of aseptic loosening consists of macrophage-mediated inflammatory responses and increasing osteoclastogenesis, which lead to an imbalance between bone formation and resorption. Despite its significant role in bone regeneration and implant osteointegration, the osteoprogenitor response to wear particles has been examined recent years. More specifically, the intracellular mechanism of osteoprogenitor mediated inflammation has not been fully elucidated. In this study, we examined the role of osteoprogenitors and the cellular mechanism by which metal wear particles elicit an inflammatory cascade. Through both in vivo and in vitro experiments, we have demonstrated that osteoprogenitor cells are capable of initiating inflammatory responses by phagocytosing wear particles, which lead to subsequent accumulation of macrophages and osteoclastogenesis, and the ERK_CEBP/β intracellular signaling is a key inflammatory pathway that links phagocytosis of wear particles to inflammatory gene expression in osteoprogenitors. AZD6244 treatment, a potent inhibitor of the ERK pathway, attenuated particle mediated inflammatory osteolysis both in vivo and in vitro. This study advances our understanding of the mechanisms of osteoprogenitor-mediated inflammation, and provides further evidence that the ERK_CEBP/β pathway may be a suitable therapeutic target in the treatment of inflammatory osteolysis. PMID:21899882

  20. 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

  1. Acetyl-L-carnitine-mediated neuroprotection during hypoxia is attributed to ERK1/2-Nrf2-regulated mitochondrial biosynthesis.

    PubMed

    Hota, Kalpana Barhwal; Hota, Sunil Kumar; Chaurasia, Om Prakash; Singh, Shashi Bala

    2012-04-01

    Neuronal damage in hypoxia and several neurodegenerative disorders is invariably associated with oxidative damage and mitochondrial dysfunction. Administration of acetyl-L-carnitine (ALCAR) on the other hand attenuates neuronal damage, prevents apoptosis, and improves energy status in hypoxic stress through less understood mechanisms. Becasue mitochondrial biogenesis could be a possible mechanism for ALCAR-induced improvement in bioenergetics in neurons, the present study aimed at exploring signaling pathways of ALCAR-induced neuroprotection in hypoxia and possible occurrence of mitochondrial biogenesis. To create global hypoxia, adult Sprague-Dawley rats were exposed to a simulated altitude of 7,620 m at standard temperature and humidity conditions. We here demonstrate that administration of ALCAR to hypoxic rats for a period of 2 weeks effectively protected hippocampal neurons from mitochondrial dysfunction, excitotoxicity, and neurodegeneration. ALCAR administration resulted in peroxisome proliferator-activated receptor γ coactivator-1α and nuclear respiratory factor-1-induced mitochondrial biogenesis, the expression of which was regulated by an extracellular-related kinase-nuclear factor erythroid 2-related factor 2 (ERK-Nrf2)-mediated mechanism. Most notably, calcium buffering into nonfunctional mitochondria ameliorated excitotoxicity and improved bioenergetic status of the hippocampal neurons. Together, the data reveal the immense therapeutic potential of ALCAR for the treatment of ischemia, stroke, and other neurodegenerative disorders associated with hypoxic stress and excitotoxicity. PMID:21542052

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  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

    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.

  8. 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

  9. 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.

  10. Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

    2015-10-01

    Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC. PMID:26212550

  11. Naringenin-induced migration of embrynoic trophectoderm cells is mediated via PI3K/AKT and ERK1/2 MAPK signaling cascades.

    PubMed

    Lim, Whasun; Song, Gwonhwa

    2016-06-15

    For successful pregnancy, a well-coordinated network of growth factors, nutrients and hormones is required for fetal-maternal interactions. Naringenin, as a weak phytoestrogen, improves diabetes, inflammation, neuronal diseases, cardiovascular diseases and cancers. However, the role of naringenin in migration mechanism(s) of peri-implantation conceptuses is unknown. Therefore, in the present study, we determined the effects of naringenin on migration of porcine trophectoderm (pTr) cells, which is a known in vitro model for research on trophectoderm cell biology and placental-fetal developmental biology, in order to assess intracellular signal transduction pathways activated by naringenin. Migration of pTr cells increased in a dose-dependent manner in response to naringenin. Also, naringenin activated the phosphorylation of AKT and ERK1/2 proteins in a dose-dependent manner and those proteins were abundant mainly in the cytoplasm of naringenin-treated pTr cells. Within 30 min after treatment with 20 μM naringenin, the abundance of phosphorylated EKR1/2, P70S6K, P90RSK and S6K proteins increased, and then returned to basal levels by 120 min whereas the abundance of AKT increased gradually to 120 min post-treatment. However, the phosphorylation of AKT, P70S6K, P90RSK and S6K was reduced in naringenin-induced pTr cells pre-treated with a PI3K inhibitor (LY294002). Also, a MEK1/2 inhibitor (U0126) significantly decreased naringenin-induced phosphorylation of ERK1/2, P70S6K and S6K proteins in pTr cells. Moreover, the naringenin-stimulated migration of pTr cells was suppressed by LY294002 and U0126. Collectively, results of the present study suggest that naringenin supports migration of pTr cells through PI3K/AKT and ERK1/2 MAPK signaling pathways crucial for orchestrating conceptus-uterine interactions. PMID:26994515

  12. 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

  13. 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

  14. 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

  15. GYF-17, a chloride substituted 2-(2-phenethyl)-chromone, suppresses LPS-induced inflammatory mediator production in RAW264.7 cells by inhibiting STAT1/3 and ERK1/2 signaling pathways.

    PubMed

    Zhu, Zhixiang; Gu, Yufan; Zhao, Yunfang; Song, Yuelin; Li, Jun; Tu, Pengfei

    2016-06-01

    GYF-17, a 2-(2-phenethyl)-chromone derivative, was isolated from agarwood and showed superior activity of inhibiting NO production of RAW264.7 cells induced by LPS in our preliminary pharmacodynamic screening. In order to develop novel therapeutic drug for acute and chronic inflammatory disorders, the anti-inflammatory activity and underlying mechanism of GYF-17 were investigated in LPS-induced RAW264.7 cells. The results showed that GYF-17 could reduce LPS-induced expression of iNOS and then result in the decrement of NO production. More meaningful, the expression and secretion of key pro-inflammatory factors, including TNF-α, IL-6 and IL-1β, were intensively inhibited by GYF-17. Furthermore, GYF-17 also down regulated the expression of COX2 and the production of PGE2 which plays important role in causing algesthesia during inflammatory response. In mechanism study, GYF-17 selectively suppressed phosphorylation of STAT1/3 and ERK1/2 during the activation of NF-κB, MAPK and STAT signaling pathways induced by LPS. Collectively, GYF-17 can intensively suppress the production of LPS-induced inflammatory mediators in RAW264.7 cells by inhibiting STAT1/3 and ERK1/2 signaling pathways and thereby shows great potential to be developed into therapeutic drug for inflammatory diseases. PMID:27064545

  16. Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis.

    PubMed

    Lin, Ming-Wei; Lin, An-Shen; Wu, Deng-Chyang; Wang, Sophie S W; Chang, Fang-Rong; Wu, Yang-Chang; Huang, Yaw-Bin

    2012-12-01

    Gastric cancer is one of the most common malignancies worldwide, and the main cause of cancer-related death in Asia. The present study assessed the anticancer effects of euphol, a triterpene alcohol with anti-inflammatory and antiviral activities on human gastric cancer cells. Euphol showed higher cytotoxicity activity against human gastric CS12 cancer cells than against noncancer CSN cells. In addition, it up-regulated the pro-apoptotic protein BAX and down-regulated the prosurvival protein Bcl-2, causing mitochondrial dysfunction, possibly by caspase-3 activation. The anti-proliferative effects of euphol were associated with the increased p27(kip1) levels and decreased cyclin B1 levels. Inhibition of ERK1/2 activation by PD98059 reversed euphol-induced pro-apoptotic protein expression and cell death. Taken together, these findings suggest that euphol selectively induced gastric cancer cells apoptosis by modulation of ERK signaling, and could thus be of value for cancer therapy. PMID:22634261

  17. 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

  18. Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

    PubMed Central

    Mediero, Aránzazu; Perez-Aso, Miguel; Cronstein, Bruce N

    2013-01-01

    Background and Purpose We previously reported that adenosine, acting at adenosine A2A receptors (A2AR), inhibits osteoclast (OC) differentiation in vitro (A2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2AR signalling. Experimental Approach OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A2AR agonist CGS21680, the A2AR antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR. Key Results A2AR stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. Conclusions and Implications A2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis. PMID:23647065

  19. 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

  20. Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons

    PubMed Central

    Borges, Gisela Patrícia; Micó, Juan Antonio; Neto, Fani Lourença

    2015-01-01

    Background: The corticotropin-releasing factor is a stress-related neuropeptide that modulates locus coeruleus activity. As locus coeruleus has been involved in pain and stress-related patologies, we tested whether the pain-induced anxiety is a result of the corticotropin-releasing factor released in the locus coeruleus. Methods: Complete Freund’s adjuvant-induced monoarthritis was used as inflammatory chronic pain model. α-Helical corticotropin-releasing factor receptor antagonist was microinjected into the contralateral locus coeruleus of 4-week-old monoarthritic animals. The nociceptive and anxiety-like behaviors, as well as phosphorylated extracellular signal-regulated kinases 1/2 and corticotropin-releasing factor receptors expression, were quantified in the paraventricular nucleus and locus coeruleus. Results: Monoarthritic rats manifested anxiety and increased phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus and paraventricular nucleus, although the expression of corticotropin-releasing factor receptors was unaltered. α-Helical corticotropin-releasing factor antagonist administration reversed both the anxiogenic-like behavior and the phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus. Conclusions: Pain-induced anxiety is mediated by corticotropin-releasing factor neurotransmission in the locus coeruleus through extracellular signal-regulated kinases 1/2 signaling cascade. PMID:25716783

  1. 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. PMID:26301823

  2. Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways.

    PubMed

    Hsieh, Cheng-Pu; Chiou, Ya-Ling; Lin, Ching-Yuang

    2010-12-01

    We investigated whether the hyperbaric oxygen (O₂) could promote the proliferation of growth-arrested osteoblasts in vitro and the mechanisms involved in this process. Osteoblasts were exposed to different combinations of saturation and pressure of O₂ and evaluated at 3 and 7 days. Control cells were cultured under ambient O₂ and normal pressure [1 atmosphere (ATA)]; high-pressure group cells were treated with high pressure (2.5 ATA) twice daily; high-O₂ group cells were treated with a high concentration O₂ (50% O₂) twice daily; and high pressure plus high-O₂ group cells were treated with high pressure (2.5 ATA) and a high concentration O₂ (50% O₂) twice daily. Hyperbaric O₂ significantly promoted osteoblast proliferation and cell cycle progression after 3 days of treatment. Hyperbaric O₂ treatment stimulated significantly increased mRNA expression of fibroblast growth factor (FGF)-2 as well as protein expression levels of Akt, p70(S6K), phosphorylated ERK, nuclear factor (NF)-κB, protein kinase C (PKC)α, and phosphorylated c-Jun N-terminal kinase (JNK). Our findings indicate that high pressure and high O₂ saturation stimulates growth-arrested osteoblasts to proliferate. These findings suggest that the proliferative effects of hyperbaric O₂ on osteoblasts may contribute to the recruitment of osteoblasts at the fracture site. The FGF-2/MEK/ERK 1/2/Akt/p70(S6K)/NF-κB and PKC/JNK pathways may be involved in mediating this process. PMID:20497028

  3. 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.

  4. Electroacupuncture-like stimulation at Baihui and Dazhui acupoints exerts neuroprotective effects through activation of the brain-derived neurotrophic factor-mediated MEK1/2/ERK1/2/p90RSK/bad signaling pathway in mild transient focal cerebral ischemia in rats

    PubMed Central

    2014-01-01

    Background This study was designed to evaluate the effects of electroacupuncture-like stimulation at Baihui (GV20) and Dazhui (GV14) acupoints (EA at acupoints) following mild cerebral ischemia-reperfusion (I/R) injury. Furthermore, we investigated whether brain-derived neurotrophic factor (BDNF)-mediated activation of extracellular signal-regulated kinase (ERK)1/2 signaling pathway is involved in the neuroprotection induced by EA at acupoints. Methods Rats were subjected to middle cerebral artery occlusion (MCAo) for 15 min followed by reperfusion for 3 d. EA at acupoints was applied 1 d postreperfusion then once daily for 2 consecutive days. Results Following the application of EA at acupoints, initiated 1 d postreperfusion, we observed significant reductions in the cerebral infarct area, neurological deficit scores, active caspase-3 protein expression, and apoptosis in the ischemic cortex after 3 d of reperfusion. We also observed markedly upregulated BDNF, phospho-Raf-1 (pRaf-1), phospho-MEK1/2 (pMEK1/2), phospho-ERK1/2 (pERK1/2), phospho-90 kDa ribosomal S6 kinase (pp90RSK), and phospho-Bad (pBad) expression, and restored neuronal nuclear antigen (NeuN) expression. Pretreatment with the MEK1/2 inhibitor U0126 abrogated the effects of EA at acupoints on cerebral infarct size, neurological deficits, active caspase-3 protein, and apoptosis in the ischemic cortex after 3 d of reperfusion. Pretreatment with U0126 also abrogated the effects of EA at acupoints on pMEK1/2, pERK1/2, pp90RSK, pBad, and NeuN expression, but did not influence BDNF and pRaf-1 expression. Conclusion Overall, our study results indicated that EA at acupoints, initiated 1 d postreperfusion, upregulates BDNF expression to provide BDNF-mediated neuroprotection against caspase-3-dependent neuronal apoptosis through activation of the Raf-1/MEK1/2/ERK1/2/p90RSK/Bad signaling cascade after 3 d of reperfusion in mild MCAo. PMID:24606810

  5. 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

  6. Selective Inhibition of MAPK Phosphatases by Zinc Accounts for ERK1/2-dependent Oxidative Neuronal Cell Death

    PubMed Central

    Ho, Yeung; Samarasinghe, Ranmal; Knoch, Megan E.; Lewis, Marcia; Aizenman, Elias; DeFranco, Donald B.

    2008-01-01

    Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinases-1/2 (ERK1/2), which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e. for Mitogen activated protein kinase [MAPK] Phosphatase-1) or as shown here by reductions in enzymatic activity (i.e. for Protein Phosphatase type 2A [PP-2A]). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress as phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole (BHA). This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death. PMID:18635668

  7. 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 Central

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

    2015-01-01

    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. PMID:26556867

  8. EGFR-Mediated Reactivation of MAPK Signaling Induces Acquired Resistance to GSK2118436 in BRAF V600E-Mutant NSCLC Cell Lines.

    PubMed

    Kim, Sung-Moo; Kim, Hwan; Jang, Kang Won; Kim, Min Hwan; Sohn, Jinyoung; Yun, Mi Ran; Kang, Han Na; Kang, Chan Woo; Kim, Hye Ryun; Lim, Sun Min; Moon, Yong Wha; Kim, Joo Hang; Paik, Soonmyung; Cho, Byoung Chul

    2016-07-01

    Although treatment of BRAF V600E-mutant non-small cell lung cancer (NSCLC(V600E)) with GSK2118436 has shown an encouraging efficacy, most patients develop resistance. To investigate the mechanisms of acquired resistance to GSK2118436 in NSCLC(V600E), we established GSK2118436-resistant (GSR) cells by exposing MV522 NSCLC(V600E) to increasing GSK2118436 concentrations. GSR cells displayed activated EGFR-RAS-CRAF signaling with upregulated EGFR ligands and sustained activation of ERK1/2, but not MEK1/2, in the presence of GSK2118436. Treatment of GSR cells with GSK2118436 enhanced EGFR-mediated RAS activity, leading to the formation of BRAF-CRAF dimers and transactivation of CRAF. Interestingly, sustained activation of ERK1/2 was partly dependent on receptor-interacting protein kinase-2 (RIP2) activity, but not on MEK1/2 activity. Combined BRAF and EGFR inhibition blocked reactivation of ERK signaling and improved efficacy in vitro and in vivo Our findings support the evaluation of combined BRAF and EGFR inhibition in NSCLC(V600E) with acquired resistance to BRAF inhibitors. Mol Cancer Ther; 15(7); 1627-36. ©2016 AACR. PMID:27196768

  9. Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

    PubMed

    de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

    2015-10-01

    are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR. Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis. PMID:26219504

  10. Lyn is involved in CD24-induced ERK1/2 activation in colorectal cancer

    PubMed Central

    2012-01-01

    Background and aim CD24 expression is associated with human colorectal cancer (CRC). Our previous data indicated that CD24 promoted the proliferation and invasion of colorectal cancer cells through the activation of ERK1/2. Since Src family kinases are frequently deregulated in CRC and closely related to the MAPK signaling pathway, we investigated the impact of Lyn, an important member of SFKs, on CD24-induced ERK1/2 activation in CRC. Methods and Results The interaction of CD24 and Lyn was identified by co-immunoprecipitation (Co-IP) and ectopic expression of CD24-induced Lyn activation. Inhibition of Lyn activation by phosphatase PP2 in SW480CD24cells abrogated CD24-induced invasion. The results of the Co-IP and immunofluorescence assay revealed that overexpression of CD24 enhanced the interaction of Lyn and ERK1/2 and induced the nuclear translocation of Lyn. However, inhibition of Lyn activity attenuated CD24-induced ERK1/2 activation, and depletion of CD24 disrupted Lyn-ERK1/2 interaction. Immunohistochemistry analysis for 202 cases of CRC showed that the expression of both CD24 and Lyn was positively correlated with tumor grade, stage, lymph node and distant metastasis. Patients with lower expression of CD24 or Lyn had a higher survival rate. The Cox multivariate analysis showed that CD24 expression, but not Lyn expression, was an independent prognostic factor of CRC. Conclusions Our results suggest that Lyn is involved in CD24-induced ERK1/2 activation in CRC. The expression of CD24 is associated with activation of Lyn and ERK1/2, which might be a novel mechanism related to CD24-mediated regulation of CRC development. PMID:22731636

  11. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation

    PubMed Central

    Kumar, Suresh M.; Liu, Shujing; Lu, Hezhe; Zhang, Hongtao; Zhang, Paul J.; Gimotty, Phyllis A.; Guerra, Matthew; Guo, Wei; Xu, Xiaowei

    2012-01-01

    There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression. PMID:22286766

  12. Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

    PubMed

    Sasseville, Maxime; Ritter, Lesley J; Nguyen, Thao M; Liu, Fang; Mottershead, David G; Russell, Darryl L; Gilchrist, Robert B

    2010-09-15

    Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation. PMID:20736313

  13. β-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and p38.

    PubMed

    Perez-Aso, Miguel; Flacco, Nicla; Carpena, Nuria; Montesinos, M Carmen; D'Ocon, Pilar; Ivorra, M Dolores

    2014-01-01

    β-Adrenoceptors (β-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of β-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that β-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline β-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after β-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium β-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis. PMID:24768830

  14. 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

  15. 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

  16. Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium

    SciTech Connect

    Liu Zhiwei; Yu Xinyuan; Shaikh, Zahir A.

    2008-05-01

    Cadmium (Cd), an endocrine disruptor, can induce a variety of signaling events including the activation of ERK1/2 and AKT. In this study, the involvement of estrogen receptors (ER) in these events was evaluated in three human breast caner cell lines, MCF-7, MDA-MB-231, and SK-BR-3. The Cd-induced signal activation patterns in the three cell lines mimicked those exhibited in response to 17{beta}-estradiol. Specifically, treatment of MCF-7 cells, that express ER{alpha}, ER{beta} and GPR30, to 0.5-10 {mu}M Cd for only 2.5 min resulted in transient phosphorylation of ERK1/2. Cd also triggered a gradual increase and sustained activation of AKT during the 60 min treatment period. In SK-BR-3 cells, that express only GPR30, Cd also caused a transient activation of ERK1/2, but not of AKT. In contrast, in MDA-MB-231 cells, that express only ER{beta}, Cd was unable to cause rapid activation of either ERK1/2 or AKT. A transient phosphorylation of ER{alpha} was also observed within 2.5 min of Cd exposure in the MCF-7 cells. While the estrogen receptor antagonist, ICI 182,780, did not prevent the effect of Cd on these signals, specific siRNA against hER{alpha} significantly reduced Cd-induced activation of ERK1/2 and completely blocked the activation of AKT. It is concluded that Cd, like estradiol, can cause rapid activation of ERK1/2 and AKT and that these signaling events are mediated by possible interaction with membrane ER{alpha} and GPR30, but not ER{beta}.

  17. Regulation of Erk1/2 activation by osteopontin in PC3 human prostate cancer cells

    PubMed Central

    2010-01-01

    Background Osteopontin (OPN) has been shown to play many roles in the progression of cancer. We have recently demonstrated the activation of Akt by OPN. Integrin-linked kinase and PI3-kinase are integral proteins in OPN/AKT pathway in PC3 cells. To investigate the role of the extracellular receptors in OPN signaling, we have examined the spatio-temporal regulation of CD44 and integrin αvβ3 receptor in OPN-induced Akt activation in PC3 cells. Results Here, our studies demonstrate that OPN can activate Akt either through the αVβ3 integrin or the CD44 cell surface receptor. Members of the Mitogen Activated Protein Kinase (MAPK) family have been shown to be up-regulated in a variety of human cancers and have been implicated in the metastatic behavior. Our studies have demonstrated an increase in the phosphorylation of c-Raf at Ser259 and Ser338 in PC3 cells over-expressing OPN. This increase matches up with the Erk1/2 phosphorylation at Thr202/204 and activation. However, the inhibition of Akt activity augments the phosphorylation state of ERK1/2 to two to three fold with a concomitant reduction in the phosphorylation state of c-Raf at Ser259. Conclusions Regulation c-Raf phosphorylation at Ser259 has a role in the anti-apoptotic pathways mediated by Akt or Raf/MEK/ERK proteins. OPN may have dual effects in the activation of Erk1/2. We propose this based on the observations that while OPN activates c-Raf and Erk1/2; it also acts to inhibit c-Raf and Erk1/2 activation through Akt pathway. Our observations suggest that the activation of c-Raf-ERK cascade may promote cell cycle arrest in prostate cancer cells and OPN signaling has a role in the anti-apoptotic mechanism. PMID:20868520

  18. Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

    SciTech Connect

    Holmstroem, Therese E.; Mattsson, Charlotte L.; Wang, Yanling; Iakovleva, Irina; Petrovic, Natasa; Nedergaard, Jan

    2010-10-01

    In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G{sub i}-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.

  19. Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator.

    PubMed

    Voong, Lilien N; Slater, Allison R; Kratovac, Sebila; Cressman, Drew E

    2008-04-01

    The expression of major histocompatibility class II genes is necessary for proper antigen presentation and induction of an immune response. This expression is initiated by the class II transactivator, CIITA. The establishment of the active form of CIITA is controlled by a series of post-translational events, including GTP binding, ubiquitination, and dimerization. However, the role of phosphorylation is less clearly defined as are the consequences of phosphorylation on CIITA activity and the identity of the kinases involved. In this study we show that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) interact directly with CIITA, targeting serine residues in the amino terminus of the protein, including serine 288. Inhibition of this phosphorylation by dominant-negative forms of ERK or by treatment of cells with the ERK inhibitor PD98059 resulted in the increase in CIITA-mediated gene expression from a class II promoter, enhanced the nuclear concentration of CIITA, and impaired its ability to bind to the nuclear export factor, CRM1. In contrast, inhibition of ERK1/2 activity had little effect on serine-to-alanine mutant forms of CIITA. These data suggest a model whereby ERK1/2-mediated phosphorylation of CIITA down-regulates CIITA activity by priming it for nuclear export, thus providing a means for cells to tightly regulate the extent of antigen presentation. PMID:18245089

  20. Estradiol and angiotensin II crosstalk in hydromineral balance: Role of the ERK1/2 and JNK signaling pathways.

    PubMed

    Almeida-Pereira, G; Coletti, R; Mecawi, A S; Reis, L C; Elias, L L K; Antunes-Rodrigues, J

    2016-05-13

    The angiotensin II (ANGII) receptor AT1 plays an important role in the control of hydromineral balance, mediating the dipsogenic and natriorexigenic effects and neuroendocrine responses of ANGII. While estradiol (E2) is known to modulate several actions of ANGII in the brain, the molecular and cellular mechanisms of the interaction between E2 and ANGII and its physiological role in the control of body fluids remain unclear. We investigated the influence of E2 (40 μg/kg) pretreatment and extracellular-signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) cell signaling on the dipsogenic and natriorexigenic effects, as well as the neuroendocrine responses to angiotensinergic central stimulation in ovariectomized rats (OVX). We showed that the inhibitory effect of E2 on ANGII-induced water and sodium intake requires the ERK1/2 and JNK signaling pathways. On the other hand, E2 pretreatment prevents the ANGII-induced phosphorylation of ERK and JNK in the lamina terminalis. E2 therapy decreased oxytocin (OT) and vasopressin (AVP) secretion and decreased ERK1/2 phosphorylation in the supraoptic and paraventricular nuclei (SON and PVN, respectively). We found that the AVP secretion induced by ANGII required ERK1/2 signaling, but OT secretion did not involve ERK1/2 signaling. Taken together, these results demonstrate that E2 modulates ANGII-induced water and sodium intake and AVP secretion by affecting the ERK1/2 and JNK pathways in the lamina terminalis and ERK1/2 signaling in the hypothalamic nuclei (PVN and SON) in OVX rats. PMID:26951941

  1. Activation of ERK1/2 Ameliorates Liver Steatosis in Leptin Receptor-Deficient (db/db) Mice via Stimulating ATG7-Dependent Autophagy.

    PubMed

    Xiao, Yuzhong; Liu, Hao; Yu, Junjie; Zhao, Zilong; Xiao, Fei; Xia, Tingting; Wang, Chunxia; Li, Kai; Deng, Jiali; Guo, Yajie; Chen, Shanghai; Chen, Yan; Guo, Feifan

    2016-02-01

    Although numerous functions of extracellular signal-regulated kinase 1/2 (ERK1/2) are identified, a direct effect of ERK1/2 on liver steatosis has not been reported. Here, we show that ERK1/2 activity is compromised in livers of leptin receptor-deficient (db/db) mice. Adenovirus-mediated activation of mitogen-activated protein kinase kinase 1 (MEK1), the upstream regulator of ERK1/2, significantly ameliorated liver steatosis in db/db mice, increased expression of genes related to fatty acid β-oxidation and triglyceride (TG) export and increased serum β-hydroxybutyrate (3-HB) levels. Opposite effects were observed in adenovirus-mediated ERK1/2 knockdown C57/B6J wild-type mice. Furthermore, autophagy and autophagy-related protein 7 (ATG7) expression were decreased or increased by ERK1/2 knockdown or activation, respectively, in primary hepatocytes and liver. Blockade of autophagy by the autophagy inhibitor chloroquine or adenovirus-mediated ATG7 knockdown reversed the ameliorated liver steatosis in recombinant adenoviruses construct expressing rat constitutively active MEK1 Ad-CA MEK1 db/db mice, decreased expression of genes related to fatty acid β-oxidation and TG export, and decreased serum 3-HB levels. Finally, ERK1/2 regulated ATG7 expression in a p38-dependent pathway. Taken together, these results identify a novel beneficial role for ERK1/2 in liver steatosis via promoting ATG7-dependent autophagy, which provides new insights into the mechanisms underlying liver steatosis and important hints for targeting ERK1/2 in treating liver steatosis. PMID:26581593

  2. Regulation of ERK1/2 mitogen-activated protein kinase by NMDA-receptor-induced seizure activity in cortical slices.

    PubMed

    Yamagata, Yoko; Kaneko, Koichi; Kase, Daisuke; Ishihara, Hiromi; Nairn, Angus C; Obata, Kunihiko; Imoto, Keiji

    2013-04-24

    Extracellular signal-regulated kinase 1/2 (ERK1/2) that belongs to a subfamily of mitogen-activated protein kinases (MAPKs) plays diverse roles in the central nervous system. Activation of ERK1/2 has been observed in various types of neuronal excitation, including seizure activity in vivo and in vitro, as well as in NMDA-receptor (NMDA-R)-dependent long-term potentiation in the hippocampus. On the other hand, recent studies in cultured neurons have shown that NMDA-R stimulation could result in either ERK1/2 activation or non-activation, depending on the pharmacological manipulations. To assess NMDA-R-dependent regulation of ERK1/2 activity in vivo, here we examined the effect of NMDA-R-induced seizure activity on ERK1/2 activation by using rat cortical slice preparations. NMDA-R-dependent seizure activity introduced by Mg2+ -free condition did not cause ERK1/2 activation. On the other hand, when picrotoxin was added to concurrently suppress GABAA-receptor-mediated inhibition, profound ERK1/2 activation occurred, which was accompanied by strong phospho-ERK1/2-staining in the superficial and deep cortical layer neurons. In this case, prolonged membrane depolarization and enhanced burst action potential firings, both of which were much greater than those in Mg2+ -free condition alone, were observed. Differential ERK1/2 activation was supported by the concurrent selective increase in phosphorylation of a substrate protein, phospho-site 4/5 of synapsin I. These results indicate that NMDA-R activation through a release from Mg2+ -blockade, which accompanies enhancement of both excitatory and inhibitory synaptic transmission, was not enough, but concurrent suppression of GABAergic inhibition, which leads to a selective increase in excitatory synaptic transmission, was necessary for robust ERK1/2 activation to occur within the cortical network. PMID:23419897

  3. Regulation of ERK1/2 mitogen-activated protein kinase by NMDA-receptor-induced seizure activity in cortical slices

    PubMed Central

    Yamagata, Yoko; Kaneko, Koichi; Kase, Daisuke; Ishihara, Hiromi; Nairn, Angus C.; Obata, Kunihiko; Imoto, Keiji

    2013-01-01

    Extracellular signal-regulated kinase 1/2 (ERK1/2) that belongs to a subfamily of mitogen-activated protein kinases (MAPKs) plays diverse roles in the central nervous system. Activation of ERK1/2 has been observed in various types of neuronal excitation, including seizure activity in vivo and in vitro, as well as in NMDA-receptor (NMDA-R)-dependent long-term potentiation in the hippocampus. On the other hand, recent studies in cultured neurons have shown that NMDA-R stimulation could result in either ERK1/2 activation or non-activation, depending on the pharmacological manipulations. To assess NMDA-R-dependent regulation of ERK1/2 activity in vivo, here we examined the effect of NMDA-R-induced seizure activity on ERK1/2 activation by using rat cortical slice preparations. NMDA-R-dependent seizure activity introduced by Mg2+-free condition did not cause ERK1/2 activation. On the other hand, when picrotoxin was added to concurrently suppress GABAA-receptor-mediated inhibition, profound ERK1/2 activation occurred, which was accompanied by strong phospho-ERK1/2-staining in the superficial and deep cortical layer neurons. In this case, prolonged membrane depolarization and enhanced burst action potential firings, both of which were much greater than those in Mg2+-free condition alone, were observed. Differential ERK1/2 activation was supported by the concurrent selective increase in phosphorylation of a substrate protein, phospho-site 4/5 of synapsin I. These results indicate that NMDA-R activation through a release from Mg2+-blockade, which accompanies enhancement of both excitatory and inhibitory synaptic transmission, was not enough, but concurrent suppression of GABAergic inhibition, which leads to a selective increase in excitatory synaptic transmission, was necessary for robust ERK1/2 activation to occur within the cortical network. PMID:23419897

  4. The EDA-containing cellular fibronectin induces epithelial-mesenchymal transition in lung cancer cells through integrin α9β1-mediated activation of PI3-K/AKT and Erk1/2.

    PubMed

    Sun, Xiaojuan; Fa, Pingping; Cui, Zhiwen; Xia, Ye; Sun, Liang; Li, Zesong; Tang, Aifa; Gui, Yaoting; Cai, Zhiming

    2014-01-01

    Cellular fibronectin (cFN) is one of the main components of tissue extracellular matrices and is involved in multiple physiologic and pathologic processes such as embryogenesis, wound healing, inflammation and tumor progression. The function of fibronectin in regulating normal cell adhesion and migration is well documented, but its function in cancer progression is only partially unraveled. We have reported previously that fibronectin stimulates the proliferation and survival of non-small lung carcinoma cells through upregulation of pro-oncogenic signals related to cyclooxygenase-2/phosphatidylinositol-3-kinase/protein kinase B (COX-2/PI3-K/AKT)/mammalian target of rapamycin triggered by activation of the integrin α5β1. Here, we extend these studies by showing that fibronectin promotes epithelial-mesenchymal transition (EMT) in lung cancer cells. We found that cFN, but not plasma fibronectin or type 1 collagen, induces lung carcinoma cell scattering in vitro, promotes cell migration and invasion of Matrigel and stimulates the expression of the mesenchymal marker α-smooth muscle actin while decreasing the expression of the epithelial marker E-cadherin through PI3-K and Erk pathways. Interestingly, the extra domain A (EDA) within cFN was found to be crucial for this process, as confirmed by testing cells overexpressing EDA or cells exposed to EDA-containing matrices. We found that the integrin α9, but not α5, mediated cFN-induced EMT as silencing integrin α9 neutralized cFN-induced EMT. Overall, our findings show that the EDA domain within cFN induces EMT in lung carcinoma cells through integrin α9-mediated activation of PI3-K and Erk. PMID:23929437

  5. 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. PMID:27301697

  6. RHAMM Promotes Interphase Microtubule Instability and Mitotic Spindle Integrity through MEK1/ERK1/2 Activity*

    PubMed Central

    Tolg, Cornelia; Hamilton, Sara R.; Morningstar, Lyndsey; Zhang, Jing; Zhang, S.; Esguerra, Kenneth V.; Telmer, Patrick G.; Luyt, Len G.; Harrison, Rene; McCarthy, James B.; Turley, Eva A.

    2010-01-01

    An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMMΔ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMMΔ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMMΔ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMMΔ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMMΔ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMMΔ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMMΔ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMMΔ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates. PMID:20558733

  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 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

  9. Role of ERK1/2 MAPK Signaling in the Maintenance of Myelin and Axonal Integrity in the Adult CNS

    PubMed Central

    Ishii, Akihiro; Furusho, Miki; Dupree, Jeffrey L.

    2014-01-01

    Oligodendrocytes form myelin during postnatal development and then maintain a functional myelin sheath throughout adult life. While many regulators of developmental myelination have been identified, the signal transduction mechanisms that regulate oligodendrocyte functions in adulthood are not well understood. The extracellular signal-regulated kinases-1 and -2 (ERK1/2), downstream mediators of mitogen-activated protein kinases (MAPKs), have emerged as prominent regulators of myelin formation. Here, we investigated whether these signaling molecules are also required for myelin maintenance in the adult CNS. Inducible conditional ablation of Erk1/2 in oligodendrocytes of the adult CNS resulted in a downregulation of myelin gene expression. Although myelin thickness was reduced and some axons were demyelinated, the majority of axons were wrapped by intact myelin sheaths that appeared structurally normal. However, late onset of progressive axonal degeneration, accompanied by astrogliosis, microglial activation, partial loss of oligodendrocytes, and functional impairment, occurred in the adult mice lacking ERK1/2 activity. Conditional ablation of Fibroblast Growth Factor receptors-1 and -2 (FGFR1/2) in oligodendrocytes also resulted in downregulation of myelin gene expression and development of axonal degeneration as the mice aged. Further, the level of the key transcription factor myelin gene regulatory factor (Myrf) was downregulated or upregulated in mice with genetic loss or gain of ERK1/2 function, respectively. Together, our studies demonstrate that ERK1/2-MAPK signaling is required for the long-term maintenance of myelin and axonal integrity in the adult CNS and suggest that FGFR1/2 and Myrf may, in part, contribute to signaling upstream and downstream of ERK1/2 in maintaining these oligodendrocyte functions during adulthood. PMID:25429144

  10. Role of ERK1/2 MAPK signaling in the maintenance of myelin and axonal integrity in the adult CNS.

    PubMed

    Ishii, Akihiro; Furusho, Miki; Dupree, Jeffrey L; Bansal, Rashmi

    2014-11-26

    Oligodendrocytes form myelin during postnatal development and then maintain a functional myelin sheath throughout adult life. While many regulators of developmental myelination have been identified, the signal transduction mechanisms that regulate oligodendrocyte functions in adulthood are not well understood. The extracellular signal-regulated kinases-1 and -2 (ERK1/2), downstream mediators of mitogen-activated protein kinases (MAPKs), have emerged as prominent regulators of myelin formation. Here, we investigated whether these signaling molecules are also required for myelin maintenance in the adult CNS. Inducible conditional ablation of Erk1/2 in oligodendrocytes of the adult CNS resulted in a downregulation of myelin gene expression. Although myelin thickness was reduced and some axons were demyelinated, the majority of axons were wrapped by intact myelin sheaths that appeared structurally normal. However, late onset of progressive axonal degeneration, accompanied by astrogliosis, microglial activation, partial loss of oligodendrocytes, and functional impairment, occurred in the adult mice lacking ERK1/2 activity. Conditional ablation of Fibroblast Growth Factor receptors-1 and -2 (FGFR1/2) in oligodendrocytes also resulted in downregulation of myelin gene expression and development of axonal degeneration as the mice aged. Further, the level of the key transcription factor myelin gene regulatory factor (Myrf) was downregulated or upregulated in mice with genetic loss or gain of ERK1/2 function, respectively. Together, our studies demonstrate that ERK1/2-MAPK signaling is required for the long-term maintenance of myelin and axonal integrity in the adult CNS and suggest that FGFR1/2 and Myrf may, in part, contribute to signaling upstream and downstream of ERK1/2 in maintaining these oligodendrocyte functions during adulthood. PMID:25429144

  11. 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.

  12. Differential ERK1/2 Signaling and Hypertrophic Response to Endothelin-1 in Cardiomyocytes from SHR and Wistar-Kyoto Rats: A Potential Target for Combination Therapy of Hypertension

    PubMed Central

    Zhu, Li-an; Fang, Ning-yuan; Gao, Ping-jin; Jin, Xian; Wang, Hai-ya; Liu, Zhenguo

    2015-01-01

    Extracellular signal regulated kinase½ (ERK1/2) signaling is critical to endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. This study was to investigate ERK1/2 signaling and hypertrophic response to ET-1 stimulation in cardiomyocytes (CMs) from spontaneous hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Primary neonatal SHR and WKY CMs were exposed to ET-1 for up to 24 hrs. Minimal basal ERK1/2 phosphorylation was present in WKY CMs, while a significant baseline ERK1/2 phosphorylation was observed in SHR CMs. ET-1 induced a time- and dose-dependent increase in ERK1/2 phosphorylation in both SHR and WKY CMs. However, ET-1-induced ERK1/2 activation occurred much earlier with significantly higher peak phosphorylation level, and stayed elevated for longer duration in SHR CMs than that in WKY CMs. ET-1-induced hypertrophic response was more prominent in SHR CMs than that in WKY CMs as reflected by increased cell surface area, intracellular actin density, and protein synthesis. Pre-treatment with ERK1/2 phosphorylation inhibitor PD98059 completely prevented ET-1-induced ERK1/2 phosphorylation and increases in cell surface area and protein synthesis in SHR and WKY CMs. The specific PI3 kinase inhibitor LY294002 blocked ET-1-induced Akt and ERK1/2 phosphorylation, and protein synthesis in CMs. These data indicated that ERK1/2 signaling was differentially enhanced in CMs, and was associated with increased cardiac hypertrophic response to ET-1 in SHR. ET-1-induced ERK1/2 activation and cardiac hypertrophy appeared to be mediated via PI3 kinase/Akt signaling in SHR and WKY. The differential ERK1/2 activation in SHR CMs by ET-1 might represent a potential target for combination therapy of hypertension. PMID:25360842

  13. Regulation of PHLDA1 Expression by JAK2-ERK1/2-STAT3 Signaling Pathway.

    PubMed

    Lyu, Ji Hyo; Huang, Bin; Park, Dae-Weon; Baek, Suk-Hwan

    2016-02-01

    Toll-like receptor 2 (TLR2)-mediated signaling cascades and gene regulation are mainly involved in diseases, such as immunity and inflammation. In this study, microarray analysis was performed using bone marrow-derived macrophages (BMDM) and Raw 264.7 cells to identify novel proteins involved in the TLR2-mediated cellular response. We found that pleckstrin homology-like domain family, member 1 (PHLDA1) is a novel gene up-regulated by TLR2 stimulation and determined the unique signaling pathway for its expression. Treatment with TLR2 agonist Pam3 CSK4 increased mRNA, protein, and fluorescence staining of PHLDA1. Induction of PHLDA1 by TLR2 stimulation disappeared from TLR2 KO mice-derived BMDM. Among janus kinase (JAK) family members, JAK2 was involved in TLR2-stimulated PHLDA1 expression. Signal transducer and activator of transcription 3 (STAT3) also participated in PHLDA1 expression downstream of the JAK2. Interestingly, ERK1/2 was an intermediate between JAK2 and STAT3. In silico analysis revealed the presence of highly conserved γ-activated sites within mouse PHLDA1 promoter and confirmed the JAK2-STAT3 pathway is important to Pam3 CSK4 -induced PHLDA1 transcription. These findings suggest that the JAK2-ERK1/2-STAT3 pathway is an important signaling pathway for PHLDA1 expression and that these proteins may play a critical role in eliciting TLR2-mediated immune and inflammatory response. PMID:26239656

  14. 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.

  15. 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

    2016-05-01

    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. PMID:26444902

  16. 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

  17. Factor XII stimulates ERK1/2 and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis

    PubMed Central

    LaRusch, Gretchen A.; Mahdi, Fakhri; Shariat-Madar, Zia; Adams, Gregory; Sitrin, Robert G.; Zhang, Wan Ming; McCrae, Keith R.

    2010-01-01

    Factor XII (FXII) and high molecular weight kininogen (HK) mutually block each other's binding to the urokinase plasminogen activator receptor (uPAR). We investigated if FXII stimulates cells by interacting with uPAR. FXII (3-62nM) with 0.05mM Zn2+ induces extracellular signal-related kinase 1/2 (ERK1/2; mitogen-activated protein kinase 44 [MAPK44] andMAPK42) and Akt (Ser473) phosphorylation in endothelial cells. FXII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. An uPAR signaling region for FXII is on domain 2 adjacent to uPAR's integrin binding site. Cleaved HK or peptides from HK's domain 5 blocks FXII-induced ERK1/2 and Akt phosphorylation. A β1 integrin peptide that binds uPAR, antibody 6S6 to β1 integrin, or the epidermal growth factor receptor (EGFR) inhibitor AG1478 blocks FXII-induced phosphorylation of ERK1/2 and Akt. FXII induces endothelial cell proliferation and 5-bromo-2′deoxy-uridine incorporation. FXII stimulates aortic sprouting in normal but not uPAR-deficient mouse aorta. FXII produces angiogenesis in matrigel plugs in normal but not uPAR-deficient mice. FXII knockout mice have reduced constitutive and wound-induced blood vessel number. In sum, FXII initiates signaling mediated by uPAR, β1 integrin, and the EGFR to induce human umbilical vein endothelial cell proliferation, growth, and angiogenesis. PMID:20228268

  18. Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells

    PubMed Central

    Wauson, Eric M.; Guerra, Marcy L.; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M.

    2015-01-01

    The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that Gi is not central to either pathway. Although glucagon-like peptide 1, an agonist for a Gs-coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca2+ entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective Gq inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for Gq. Inhibition of G12/13 by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways. PMID:26168033

  19. 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. PMID:26324779

  20. 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...

  1. 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

  2. Bovine lactoferricin induces TIMP-3 via the ERK1/2-Sp1 axis in human articular chondrocytes

    PubMed Central

    Yan, Dongyao; Chen, Di; Hawse, John R; van Wijnen, Andre J; Im, Hee-Jeong

    2013-01-01

    Bovine lactoferricin (LfcinB) is a heparan sulfate-binding peptide with multiple bioactivities. In human articular cartilage, LfcinB antagonizes interleukin-1 β (IL-1β) and fibroblast growth factor 2 (FGF-2) in proteoglycan metabolism, catabolic protease expression, and induction of pro-inflammatory mediators. LfcinB specifically activates ERK1/2, p38 and Akt, but whether these signaling pathways control the expression of LfcinB target genes remained unknown. In this report, we characterized a novel aspect of LfcinB-mediated genetic response in human articular chondrocytes, tissue inhibitor of metalloproteinase 3 (TIMP-3) induction. Inhibition of individual signaling pathways revealed that ERK1/2 functions as the major pathway in TIMP-3 expression, whereas Akt plays a minor role. Further investigation identified Sp1 as a critical transcriptional activator in TIMP-3 regulation, and Sp1 activity is modulated by ERK1/2, not Akt. Comparative quantification indicates significant downregulation of TIMP-3 occurs in OA chondrocytes, suggesting a beneficial role of LfcinB in OA pathogenesis. Our results collectively provide new insights into the mechanism of action of LfcinB, and support the candidacy of LfcinB as a chondroprotective agent. PMID:23313877

  3. 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. PMID:26119854

  4. NMDA-stimulated ERK1/2 Signaling and the Transcriptional Up-regulation of Plasticity-related Genes are Developmentally Regulated following in vitro Neuronal Maturation

    PubMed Central

    Zhou, Xianju; Moon, Changjong; Zheng, Fei; Luo, Yongneng; Soellner, Deborah; Nuñez, Joseph L.; Wang, Hongbing

    2010-01-01

    The general features of neuroplasticity are developmentally regulated. Although it has been hypothesized that the loss of plasticity in mature neurons may be due to synaptic saturation and functional reduction of NMDA receptors (NMDAR), the molecular mechanisms remain largely unknown. We examined the effects of NMDAR activation and KCl-mediated membrane depolarization on ERK1/2 signaling following in vitro maturation of cultured cortical neurons. Although NMDA stimulated robust increase of intracellular calcium at both DIV (day in vitro) 3 and 14, the activation of ERK1/2 and CREB was impaired at DIV 14. Specifically, the phosphorylation of ERK1/2 was stimulated by both NMDA and KCl at DIV 3. However, at DIV 14, NMDA-, but not KCl-stimulated ERK1/2 and CREB phosphorylation was significantly diminished. Consistently, the NMDA-induced transcription of ERK/CREB-regulated genes Bdnf exon 4, Arc and zif268 was significantly attenuated at DIV 14. Moreover, compared to DIV 3 neurons, the basal level of phosphorylated-ERK1/2 in DIV 14 neurons increased tremendously following maturation, and was more susceptible to dephosphorylation. Blocking calcium channels by nifedipine or NMDAR by APV caused more dramatic ERK dephosphorylation in DIV 14 neurons. We further demonstrate that the loss of plasticity-related signaling is unrelated to NMDA-induced cell death of the DIV 14 neurons. Taken together, these results suggest that the attenuation of certain aspects of neuroplasticity following maturation may be due to the reduction of NMDAR-mediated gene transcription and a saturation of ERK1/2 activity. PMID:19396876

  5. GPCR-like signaling mediated by smoothened contributes to acquired chemoresistance through activating Gli

    PubMed Central

    2014-01-01

    Background Smoothened (Smo), which possesses a structural similarity with classic G-protein coupled receptors (GPCR), is the most important molecular target in Hedgehog (Hh) signaling system for developing anticancer drugs; however, whether Smo may transmit GPCR-like signaling to activate the canonical transcriptional factor Gli of Hh signaling system and consequently to be involved in the Gli-dependent biological events remains controversial. Results In this study, using the acquired chemoresistant cancer cell lines and their respective parental cells, we found that Smo may activate Gli through Gαi, Gβγ-JNK signaling axis, thereby promoting the Gli-dependent acquired chemoresistance. These observations were further complementarily strengthened by data obtained from chemosensitive cancer cells with artificially elevated Hh pathway activity. Conclusions Hence, our data demonstrate that GPCR-like signaling mediated by Smo contributes to the acquired chemoresistance through activating the canonical Hh transcriptional factor Gli; therefore improving our knowledge of the nature of the signal transduction of Smo and the molecular mechanisms responsible for the acquired chemoresistance maintained by Hh pathway. Moreover, our data that JNK after activated by Smo-Gβγ signaling axis may stimulate the Gli activity and consequently promotes acquired chemoresistance expose a promising and potential target for developing anti-cancer drugs aimed at Hh pathway and for combating the acquired resistance raised by using of anti-cancer drugs targeting Smo. PMID:24393163

  6. Ursolic Acid Triggers Apoptosis in Human Osteosarcoma Cells via Caspase Activation and the ERK1/2 MAPK Pathway.

    PubMed

    Wu, Chia-Chieh; Cheng, Chun-Hsiang; Lee, Yi-Hui; Chang, Ing-Lin; Chen, Hsin-Yao; Hsieh, Chen-Pu; Chueh, Pin-Ju

    2016-06-01

    Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, has been shown to trigger apoptosis in several lines of tumor cells in vitro. We found that treatment with UA suppressed the viability of human osteosarcoma MG-63 cells and induced cell cycle arrest at sub-G1 and G2/M phases. Furthermore, exposure to UA induced intracellular oxidative stress and collapse of mitochondrial membrane permeability, resulting in the subsequent activation of apoptotic caspases 8, 9, and 3 as well as PARP cleavage, and ultimately apoptosis in MG-63 cells. Moreover, protein analysis of mitogen-activated protein kinase (MAPK)-related protein expression showed an increase in activated ERK1/2, JNK, and p38 MAPK in UA-treated MG-63 cells. In addition, UA-induced apoptosis was significantly abolished in MG-63 cells that had been pretreated with inhibitors of caspase 3, 8, and 9 and ERK1/2. Furthermore, UA-treated MG-63 cells also exhibited an enhancement in Bax/Bcl-2 ratio, whereas anti-apoptotic XIAP and survivin were down-regulated. Taken together, we provide evidence demonstrating that UA mediates caspase-dependent and ERK1/2 MAPK-associated apoptosis in osteosarcoma MG-63 cells. PMID:27171502

  7. 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. PMID:27475840

  8. Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling

    PubMed Central

    Wang, Juanfei; Ren, Jinqi; Wu, Bin; Feng, Shanshan; Cai, Guoping; Tuluc, Florin; Peränen, Johan; Guo, Wei

    2015-01-01

    Exocytosis is tightly regulated in many cellular processes, from neurite expansion to tumor proliferation. Rab8, a member of the Rab family of small GTPases, plays an important role in membrane trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rabin8 is a guanine nucleotide exchange factor (GEF) and major activator of Rab8. Investigating how Rabin8 is activated in cells is thus pivotal to the understanding of the regulation of exocytosis. Here we show that phosphorylation serves as an important mechanism for Rabin8 activation. We identified Rabin8 as a direct phospho-substrate of ERK1/2 in response to EGF signaling. At the molecular level, ERK phosphorylation relieves the autoinhibition of Rabin8, thus promoting its GEF activity. We further demonstrate that blocking ERK1/2-mediated phosphorylation of Rabin8 inhibits transferrin recycling to the plasma membrane. Together, our results suggest that ERK1/2 activate Rabin8 to regulate vesicular trafficking to the plasma membrane in response to extracellular signaling. PMID:25535387

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

    PubMed

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

    2016-04-01

    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; NEST-INFM, Scuola Normale Superiore, Pisa ; Pancrazi, Laura; Maffei, Margherita; Institute of Food Science CNR, Avellino ; 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. Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

    PubMed

    He, Ze; Sun, Qin; Liang, Yuan-Jiao; Chen, Li; Ge, Yi-Feng; Yun, Shi-Feng; Yao, Bing

    2016-01-01

    This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells. PMID:26289400

  13. Activation of Toll-like receptor 3 increases mouse aortic vascular smooth muscle cell contractility through ERK1/2 pathway.

    PubMed

    Hardigan, Trevor; Spitler, Kathryn; Matsumoto, Takayuki; Carrillo-Sepulveda, Maria Alicia

    2015-11-01

    Activation of Toll-like receptor 3 (TLR3), a pattern recognition receptor of the innate immune system, is associated with vascular complications. However, whether activation of TLR3 alters vascular contractility is unknown. We, therefore, hypothesized that TLR3 activation augments vascular contractility and activates vascular smooth muscle cell (VSMC) contractile apparatus proteins. Male mice were treated with polyinosinic-polycytidylic acid (Poly I:C group, 14 days), a TLR3 agonist; control mice received saline (vehicle, 14 days). At the end of protocol, blood pressure was measured by tail cuff method. Aortas were isolated and assessed for contractility experiments using a wire myograph. Aortic protein content was used to determine phosphorylated/total interferon regulatory factor 3 (IRF3), a downstream target of TLR3 signaling, and ERK1/2 using Western blot. We investigated the TLR3/IRF3/ERK1/2 signaling pathway and contractile-related proteins such as phosphorylated/total myosin light chain (MLC) and caldesmon (CaD) in aortic VSMC primary cultures. Poly I:C-treated mice exhibited (vs. vehicle-treated mice) (1) elevated systolic blood pressure. Moreover, Poly I:C treatment (2) enhanced aortic phenylephrine-induced maximum contraction, which was suppressed by PD98059 (ERK1/2 inhibitor), and (3) increased aortic levels of phosphorylated IRF3 and ERK1/2. Stimulation of mouse aortic VSMCs with Poly I:C resulted in increased phosphorylation of IRF3, ERK1/2, MLC, and CaD. Inhibition of ERK1/2 abolished Poly I:C-mediated phosphorylation of MLC and CaD. Our data provide functional evidence for the role of TLR3 in vascular contractile events, suggesting TLR3 as a potential new therapeutic target in vascular dysfunction and regulation of blood pressure. PMID:25724934

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Upregulation of ERK1/2-eNOS via AT2 receptors decreases the contractile response to angiotensin II in resistance mesenteric arteries from obese rats.

    PubMed

    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

  19. Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes.

    PubMed

    Hdud, Ismail M; Mobasheri, Ali; Loughna, Paul T

    2014-06-01

    The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca(2+) and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P < 0.001). PMID:24671100

  20. 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

  1. 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.

  2. 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

  3. 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

  4. DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

    PubMed

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-11-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  5. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    PubMed Central

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  6. ERK1 as a Therapeutic Target for Dendritic Cell Vaccination against High-Grade Gliomas.

    PubMed

    Ku, Min-Chi; Edes, Inan; Bendix, Ivo; Pohlmann, Andreas; Waiczies, Helmar; Prozorovski, Tim; Günther, Martin; Martin, Conrad; Pagès, Gilles; Wolf, Susanne A; Kettenmann, Helmut; Uckert, Wolfgang; Niendorf, Thoralf; Waiczies, Sonia

    2016-08-01

    Glioma regression requires the recruitment of potent antitumor immune cells into the tumor microenvironment. Dendritic cells (DC) play a role in immune responses to these tumors. The fact that DC vaccines do not effectively combat high-grade gliomas, however, suggests that DCs need to be genetically modified specifically to promote their migration to tumor relevant sites. Previously, we identified extracellular signal-regulated kinase (ERK1) as a regulator of DC immunogenicity and brain autoimmunity. In the current study, we made use of modern magnetic resonance methods to study the role of ERK1 in regulating DC migration and tumor progression in a model of high-grade glioma. We found that ERK1-deficient mice are more resistant to the development of gliomas, and tumor growth in these mice is accompanied by a higher infiltration of leukocytes. ERK1-deficient DCs exhibit an increase in migration that is associated with sustained Cdc42 activation and increased expression of actin-associated cytoskeleton-organizing proteins. We also demonstrated that ERK1 deletion potentiates DC vaccination and provides a survival advantage in high-grade gliomas. Considering the therapeutic significance of these results, we propose ERK1-deleted DC vaccines as an additional means of eradicating resilient tumor cells and preventing tumor recurrence. Mol Cancer Ther; 15(8); 1975-87. ©2016 AACR. PMID:27256374

  7. ERK1/2 can feedback-regulate cellular MEK1/2 levels.

    PubMed

    Hong, Seung-Keun; Wu, Pui-Kei; Karkhanis, Mansi; Park, Jong-In

    2015-10-01

    Signal transduction of the Raf/MEK/ERK pathway is regulated by various feedback mechanisms. Given the greater molar ratio between Raf-MEK than between MEK-ERK in cells, it may be possible that MEK1/2 levels are regulated to modulate Raf/MEK/ERK activity upon pathway stimulation. Nevertheless, it has not been reported whether MEK1/2 expression can be subject to a feedback regulation. Here, we report that the Raf/MEK/ERK pathway can feedback-regulate cellular MEK1 and MEK2 levels. In different cell types, ΔRaf-1:ER- or B-Raf(V600E)-mediated MEK/ERK activation increased MEK1 but decreased MEK2 levels. These regulations were abrogated by ERK1/2 knockdown mediated by RNA interference, suggesting the presence of a feedback mechanism that regulates MEK1/2 levels. Subsequently, analyses using qPCR and luciferase reporters of the DNA promoter and 3' untranslated region revealed that the feedback MEK1 upregulation was in part attributed to increased transcription. However, the feedback MEK2 downregulation was only observed at protein levels, which was blocked by the proteasome inhibitors, MG132 and bortezomib, suggesting that the MEK2 regulation is mediated at a post-translational level. These results suggest that the Raf/MEK/ERK pathway can feedback-regulate cellular levels of MEK1 and MEK2, wherein MEK1 levels are upregulated at transcriptional level whereas MEK2 levels are downregulated at posttranslational level. PMID:26163823

  8. Prostaglandin D(2) induces apoptosis of human osteoclasts through ERK1/2 and Akt signaling pathways.

    PubMed

    Yue, Li; Haroun, Sonia; Parent, Jean-Luc; de Brum-Fernandes, Artur J

    2014-03-01

    In a recent study we have shown that prostaglandin D2 (PGD2) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cell (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD2-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinositol3-kinase (PI3K) and NF-κB/IκB kinase-2 (IKK2) prior to the treatments of PGD2 as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-κB signaling pathways. Treatments with both PGD2 and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased β-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandin production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phosphorylations, and reduced the phosphorylation of β-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD2 and a CRTH2 agonist. Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD2, whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K

  9. Bordetella pertussis Acquires Resistance to Complement-Mediated Killing In Vivo

    PubMed Central

    Pishko, Elizabeth J.; Betting, David J.; Hutter, Christina S.; Harvill, Eric T.

    2003-01-01

    In order to initially colonize a host, bacteria must avoid various components of the innate immune system, one of which is complement. The genus Bordetella includes three closely related species that differ in their ability to resist complement-mediated killing. Bordetella parapertussis and Bordetella bronchiseptica resist killing in naïve serum, a characteristic that may aid in efficient respiratory tract colonization and has been attributed to expression of O antigen. Bordetella pertussis lacks O antigen and is sensitive to naïve serum in vitro, yet it also efficiently colonizes the respiratory tract. Based on these observations, we hypothesized that B. pertussis may have an alternate mechanism to resist complement in vivo. While a number of reports on serum sensitivity of the bordetellae have been published, we show here that serum concentration and growth conditions can greatly alter the observed level of sensitivity to complement and that all but one strain of B. pertussis observed were sensitive to some level of naïve serum in vitro, particularly when there was excess complement. However, B. pertussis rapidly acquires increased resistance in vivo to naïve serum that is specific to the alternative pathway. Resistance is not efficiently acquired by B. parapertussis and B. bronchiseptica mutants lacking O antigen. This B. pertussis-specific mechanism of complement resistance does not appear to be dependent on either brkA or other genes expressed specifically in the Bvg+ phase. This in vivo acquisition of alternative pathway resistance suggests that there is a novel O antigen-independent method by which B. pertussis evades complement-mediated killing. PMID:12933835

  10. Bordetella pertussis acquires resistance to complement-mediated killing in vivo.

    PubMed

    Pishko, Elizabeth J; Betting, David J; Hutter, Christina S; Harvill, Eric T

    2003-09-01

    In order to initially colonize a host, bacteria must avoid various components of the innate immune system, one of which is complement. The genus Bordetella includes three closely related species that differ in their ability to resist complement-mediated killing. Bordetella parapertussis and Bordetella bronchiseptica resist killing in naïve serum, a characteristic that may aid in efficient respiratory tract colonization and has been attributed to expression of O antigen. Bordetella pertussis lacks O antigen and is sensitive to naïve serum in vitro, yet it also efficiently colonizes the respiratory tract. Based on these observations, we hypothesized that B. pertussis may have an alternate mechanism to resist complement in vivo. While a number of reports on serum sensitivity of the bordetellae have been published, we show here that serum concentration and growth conditions can greatly alter the observed level of sensitivity to complement and that all but one strain of B. pertussis observed were sensitive to some level of naïve serum in vitro, particularly when there was excess complement. However, B. pertussis rapidly acquires increased resistance in vivo to naïve serum that is specific to the alternative pathway. Resistance is not efficiently acquired by B. parapertussis and B. bronchiseptica mutants lacking O antigen. This B. pertussis-specific mechanism of complement resistance does not appear to be dependent on either brkA or other genes expressed specifically in the Bvg(+) phase. This in vivo acquisition of alternative pathway resistance suggests that there is a novel O antigen-independent method by which B. pertussis evades complement-mediated killing. PMID:12933835

  11. CacyBP/SIP inhibits Doxourbicin-induced apoptosis of glioma cells due to activation of ERK1/2.

    PubMed

    Tang, Yuan; Zhan, Wenjian; Cao, Tong; Tang, Tianjin; Gao, Yong; Qiu, Zhichao; Fu, Chunling; Qian, Fengyuan; Yu, Rutong; Shi, Hengliang

    2016-03-01

    Calcyclin-binding protein or Siah-1-interacting protein (CacyBP/SIP) was previously reported to promote the proliferation of glioma cells. However, the effect of CacyBP/SIP on apoptosis of glioma is poorly understood. Here, our study shows that CacyBP/SIP plays a role in inhibiting doxorubicin (DOX) induced apoptosis of glioma cells U251 and U87. Overexpression of CacyBP/SIP obviously suppressed the DOX-induced cell apoptosis. On the contrary, silencing of CacyBP/SIP significantly promoted it. Further investigation indicated that inhibition of apoptosis by CacyBP/SIP was relevant to its nuclear translocation in response to the DOX treatment. Importantly, we found that the level of p-ERK1/2 in nuclei was related to the nuclear accumulation of CacyBP/SIP. Finally, the role of CacyBP/SIP was confirmed in vivo in a mouse model with the cell line stably silencing CacyBP/SIP. Taken together, our results suggest that CacyBP/SIP plays an important role in inhibiting apoptosis of glioma cells which might be mediated by ERK1/2 signaling pathway, which will provide some guidance for the treatment of glioma. PMID:26825673

  12. TCDD promotes lung tumors via attenuation of apoptosis through activation of the Akt and ERK1/2 signaling pathways.

    PubMed

    Chen, Rong-Jane; Siao, Shih-He; Hsu, Chung-Huei; Chang, Chu-Yung; Chang, Louis W; Wu, Chih-Hsiung; Lin, Pinpin; Wang, Ying-Jan

    2014-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multiple-site, multiple-species carcinogen that induces cancer in multiple organs. The molecular mechanisms underlying TCDD-induced lung tumorigenesis remain unclear. In the present study, a two-stage lung tumorigenesis model was established by administrating a single low dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) combined with TCDD to female A/J mice. The results indicated that TCDD combined with low-dose NNK has a significant tumor-promoting effect compared with TCDD or low-dose NNK alone. Resistance to apoptosis is a hallmark of cancer and is thought to be one of the tumor-promoting mechanisms regulated by TCDD. We performed an additional series of experiments in the normal human bronchial epithelial cell line Beas2B cells, in which TCDD was combined with the apoptosis inducer staurosporine. Our in vitro results confirmed that TCDD could rescue cells from apoptosis induced by staurosporine. The inhibition of apoptosis is likely mediated by the activation of the Akt and ERK1/2 pathways, as determined by the effectiveness of pathway-specific inhibitors in abrogating the anti-apoptotic activity of TCDD. In conclusion, we demonstrated that TCDD promoted NNK-induced lung tumorigenesis and revealed that TCDD inhibits staurosporine-induced apoptosis, at least in part, through the Akt and ERK1/2 signaling pathways. PMID:24927102

  13. TCDD Promotes Lung Tumors via Attenuation of Apoptosis through Activation of the Akt and ERK1/2 Signaling Pathways

    PubMed Central

    Siao, Shih-He; Hsu, Chung-Huei; Chang, Chu-Yung; Chang, Louis W.; Lin, Pinpin; Wang, Ying-Jan

    2014-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multiple-site, multiple-species carcinogen that induces cancer in multiple organs. The molecular mechanisms underlying TCDD-induced lung tumorigenesis remain unclear. In the present study, a two-stage lung tumorigenesis model was established by administrating a single low dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) combined with TCDD to female A/J mice. The results indicated that TCDD combined with low-dose NNK has a significant tumor-promoting effect compared with TCDD or low-dose NNK alone. Resistance to apoptosis is a hallmark of cancer and is thought to be one of the tumor-promoting mechanisms regulated by TCDD. We performed an additional series of experiments in the normal human bronchial epithelial cell line Beas2B cells, in which TCDD was combined with the apoptosis inducer staurosporine. Our in vitro results confirmed that TCDD could rescue cells from apoptosis induced by staurosporine. The inhibition of apoptosis is likely mediated by the activation of the Akt and ERK1/2 pathways, as determined by the effectiveness of pathway-specific inhibitors in abrogating the anti-apoptotic activity of TCDD. In conclusion, we demonstrated that TCDD promoted NNK-induced lung tumorigenesis and revealed that TCDD inhibits staurosporine-induced apoptosis, at least in part, through the Akt and ERK1/2 signaling pathways. PMID:24927102

  14. HMGB1 binding to receptor for advanced glycation end products enhances inflammatory responses of human bronchial epithelial cells by activating p38 MAPK and ERK1/2.

    PubMed

    Liang, Yue; Hou, Changchun; Kong, Jinliang; Wen, Hanchun; Zheng, Xiaowen; Wu, Lihong; Huang, Hong; Chen, Yiqiang

    2015-07-01

    The proinflammatory factor high mobility group box protein 1 (HMGB1) has been implicated as an important mediator of many chronic inflammatory diseases, including asthma. Human bronchial epithelial cells (HBECs) play a central role in the pathogenesis of asthma. However, the effects of HMGB1 on HBECs and the underlying mechanisms remain unknown. Here, we investigated receptor expression and proinflammatory cytokine production by primary cultures of HBECs stimulated by HMGB1. We then examined the effects of specific receptor blockade and inhibition of p38 MAPK, ERK1/2, or PI3-K on HMGB1-induced expression of proinflammatory cytokines. HMGB1 increased the expression and secretion of TNF-α, TSLP, MMP-9, and VEGF in a dose- and time-dependent manner. HMGB1 also induced elevated expression of RAGE protein. Secretion of TNF-α, VEGF, MMP-9, and TSLP was significantly decreased by RAGE blockade and p38 MAPK pathway inhibition, while a less pronounced effect was mediated by ERK1/2 inhibition. These observations suggest that HMGB1 binds RAGE and promotes activities of p38 MAPK and ERK1/2 pathways in HBECs. This then enhances the expression of TNF-α, VEGF, MMP-9, and TSLP, which are the important inflammatory factors in asthma. These results demonstrate that HMGB1 enhances the inflammatory responses of HBECs, which are involved in the modulation of inflammatory processes in asthma. PMID:25862459

  15. Dual roles of ERK1/2 in cellular senescence induced by excess thymidine in HeLa cells.

    PubMed

    Kudo, Ikuru; Nozawa, Megumi; Miki, Kensuke; Takauji, Yuki; En, Atsuki; Fujii, Michihiko; Ayusawa, Dai

    2016-08-15

    DNA damage response is crucially involved in cellular senescence. We have previously shown that excess thymidine, which stalls DNA replication forks, induces cellular senescence in human cells, and ERK1/2 play a key role in the induction of it. In this study, we found that Chk1 and ERK1/2 were activated to promote cell survival upon addition of excess thymidine. Knockdown of ERK1/2 activated Chk1, and conversely, knockdown of Chk1 activated ERK1/2, which observations suggested a mechanism for compensatory activation of Chk1 and ERK1/2 in the absence of ERK1/2 and Chk1, respectively. We also found that Chk1 functioned mainly at the onset of cellular senescence, and on the other hand, ERK1/2 functioned for a more extended period to induce cellular senescence. Our findings suggested that Chk1 and ERK1/2 were activated to promote cell survival upon addition of excess thymidine, but prolonged activation of ERK1/2 led to cellular senescence. This implies a pleiotropic effect of ERK1/2 in cellular senescence induced by excess thymidine. PMID:27443255

  16. SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells

    PubMed Central

    Saloura, Vassiliki; Cho, Hyun-Soo; Suzuki, Takehiro; Dohmae, Naoshi; Alachkar, Houda; Nakamura, Yusuke; Hamamoto, Ryuji

    2015-01-01

    The oncogenic protein ERK, a member of the extracellular signal-regulated kinase (ERK) cascade, is a well characterized signaling molecule involved in tumorigenesis. The ERK signaling pathway is activated in a large proportion of cancers and plays a critical role in tumor development. Functional regulation by phosphorylation of kinases in the ERK pathway has been extensively studied, however methylation of the ERK protein has not been reported to date. Here, we demonstrated that the protein lysine methyltransferase SUV420H1 tri-methylated ERK1 at lysines 302 and 361, and that substitution of methylation sites diminished phosphorylation levels of ERK1. Concordantly, knockdown of SUV420H1 reduced phosphorylated ERK1 and total ERK1 proteins, and interestingly suppressed ERK1 at the transcriptional level. Our results indicate that overexpression of SUV420H1 may result in activation of the ERK signaling pathway through enhancement of ERK phosphorylation and transcription, thereby providing new insights in the regulation of the ERK cascade in human cancer. PMID:26586479

  17. Interaction of mTOR and Erk1/2 signaling to regulate oligodendrocyte differentiation.

    PubMed

    Dai, JinXiang; Bercury, Kathryn K; Macklin, Wendy B

    2014-12-01

    A multitude of factors regulate oligodendrocyte differentiation and remyelination, and to elucidate the mechanisms underlying this process, we analyzed the interactions of known signaling pathways involved in these processes. Previous work from our lab and others shows that Akt, mTOR, and Erk 1/2 are major signaling pathways regulating oligodendrocyte differentiation and myelination in vitro and in vivo. However, the relative contribution of the different pathways has been difficult to establish because the impact of inhibiting one pathway in in vitro cell culture models or in vivo may alter signaling through the other pathway. These studies were undertaken to clarify the interactions between these major pathways and understand more specifically the crosstalk between them. Oligodendrocyte differentiation in vitro required Akt, mTOR, and Erk 1/2 signaling, as inhibition of Akt, mTOR, or Erk 1/2 resulted in a significant decrease of myelin basic protein mRNA and protein expression. Interestingly, while inhibition of the Erk1/2 pathway had little impact on Akt/mTOR signaling, inhibition of the Akt/mTOR pathways significantly increased Erk1/2 signaling, although not enough to overcome the loss of Akt/mTOR signaling in the regulation of oligodendrocyte differentiation. Furthermore, such crosstalk was also noted in an in vivo context, after mTOR inhibition by rapamycin treatment of perinatal pups. GLIA 2014;62:2096-2109. PMID:25060812

  18. Ultraviolet Radiation and 12-O-Tetradecanoylphorbol-13-Acetate-Induced Interaction of Mouse Epidermal Protein Kinase Cε With Stat3 Involve Integration With Erk1/2

    PubMed Central

    Sand, Jordan Marshall; Hafeez, Bilal Bin; Aziz, Moammir Hasan; Siebers, Emily Marie; Dreckschmidt, Nancy Ellen; Verma, Ajit Kumar

    2012-01-01

    We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present severa novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m2/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention. PMID:21480396

  19. Ultraviolet radiation and 12-O-tetradecanoylphorbol-13-acetate-induced interaction of mouse epidermal protein kinase Cε with Stat3 involve integration with ERK1/2.

    PubMed

    Sand, Jordan Marshall; Bin Hafeez, Bilal; Aziz, Moammir Hasan; Siebers, Emily Marie; Dreckschmidt, Nancy Ellen; Verma, Ajit Kumar

    2012-04-01

    We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present several novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m(2)/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention. PMID:21480396

  20. AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

    PubMed Central

    Hong, Seung-Keun; Jeong, Joseph H.; Chan, Andrew M.; Park, Jong-In

    2013-01-01

    Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation. Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (Δ4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated BRaf regulation was also detected in a panel of PCa lines derived from the cPten-/- L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells. PMID:23701950

  1. Connexin43-containing gap junctions potentiate extracellular Ca²⁺-induced odontoblastic differentiation of human dental pulp stem cells via Erk1/2.

    PubMed

    Li, Shiting; He, Haitao; Zhang, Gang; Wang, Fei; Zhang, Ping; Tan, Yinghui

    2015-10-15

    Extracellular Ca(2+) can promote dentin sialophosphoprotein (DSPP) expression and odontoblastic differentiation of dental pulp stem cells (DPSCs). Gap junctions mediated by connexin43 (Cx43) allow diffusion of small molecules (such as Ca(2+)) among cells to regulate cell-to-cell communications. However, it is unclear whether Cx43 is required for the Ca(2+)-induced cell differentiation. Here, we found that the influx of extracellular Ca(2+) through L-type Ca(2+) channels increases intracellular free Ca(2+) levels to promote DSPP expression. Cx43 overexpression potentiated the extracellular Ca(2+)-induced DSPP expression via Erk1/2. Flow cytometry analyses showed that Cx43 increased the percentage of p-Erk1/2 positive cells in response to Ca(2+), indicating that Cx43 in DPSCs possibly acts as a traditional gap junction channel, which permits the sharing of signals among coupled cells to make more DPSCs respond to Ca(2+). Furthermore, inhibition of Cx43 function and gap junction communication decreased Ca(2+)-induced the expression of DSPP, suggesting that cell-to-cell contacts are required for Cx43 to promote the Ca(2+)-induced cell differentiation. Similarly, the study performed on DPSCs cultured at low-density and high-density revealed that intercellular contacts are required to potentiate Erk1/2 activity and DSPP expression. In total, this study indicates that Cx43 increases Ca(2+)-induced DSPP expression and odontoblastic differentiation of DPSCs via Erk1/2 through gap junction-mediated cell-to-cell contacts. PMID:26376117

  2. 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

  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. PMID:26538070

  4. Dual role of B cells in mediating innate and acquired immunity to herpes simplex virus infections.

    PubMed

    Deshpande, S P; Kumaraguru, U; Rouse, B T

    2000-06-15

    mu-immunoglobulin chain gene targeted B-cell-deficient mice of susceptible BALB/c strain and resistant C57B1/6 strain are up to 100- to 1000-fold more susceptible to cutaneous infection by herpes simplex virus (HSV) than the respective control wild type mice. The effect of the lack of B cells on immunity to HSV infections was analyzed and B cells were found to play a dual role in affecting both innate and acquired immune responses. Natural antibodies (IgM isotype), reactive with HSV have an anti-viral effect in the innate control of primary cutaneous HSV infection. B cells can also function as antigen-presenting cells for the stimulation of HSV-specific CD4+ T-cell responses. Consequently, CD4+ T cells and interferon-gamma responses were found to be significantly impaired in HSV-infected B-cell-deficient mice compared to that seen in control mice. No significant differences were found in natural-killer-cell- or HSV-specific CD8+ T-cell activity between control and B-cell-deficient mice. Our results imply a role for B cell in mediating innate and CD4+ T-cell-specific immunity in determining susceptibility to primary HSV infections. PMID:10896767

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

    PubMed

    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

  6. Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT(2B) receptor and the ERK1/2 signalling pathway.

    PubMed

    Li, Xianxian; Ma, Yuanyuan; Wu, Xiangnan; Hao, Zhichao; Yin, Jian; Shen, Jiefei; Li, Xiaoyu; Zhang, Ping; Wang, Hang

    2013-11-29

    Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine. PMID:24211588

  7. Epidermal growth factor and perlecan fragments produced by apoptotic endothelial cells co-ordinately activate ERK1/2-dependent antiapoptotic pathways in mesenchymal stem cells.

    PubMed

    Soulez, Mathilde; Sirois, Isabelle; Brassard, Nathalie; Raymond, Marc-André; Nicodème, Frédéric; Noiseux, Nicolas; Durocher, Yves; Pshezhetsky, Alexei V; Hébert, Marie-Josée

    2010-04-01

    Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum-free medium conditioned by apoptotic EC increases extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl-xL protein levels and poly (ADP-ribose) polymerase cleavage) of human MSC. A C-terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with beta1-integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering beta1-integrin and EGF receptor-dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response. PMID:20201065

  8. 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

  9. 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

  10. Reduced dosage of ERF causes complex craniosynostosis in humans and mice, and links ERK1/2 signaling to regulation of osteogenesis

    PubMed Central

    Twigg, Stephen R F; Vorgia, Elena; McGowan, Simon J; Peraki, Ioanna; Fenwick, Aimée L; Sharma, Vikram P; Allegra, Maryline; Zaragkoulias, Andreas; Akha, Elham Sadighi; Knight, Samantha J L; Lord, Helen; Lester, Tracy; Izatt, Louise; Lampe, Anne K; Mohammed, Shehla N; Stewart, Fiona J; Verloes, Alain; Wilson, Louise C; Healy, Chris; Sharpe, Paul T; Hammond, Peter; Hughes, Jim; Taylor, Stephen; Johnson, David; Wall, Steven A; Mavrothalassitis, George; Wilkie, Andrew O M

    2013-01-01

    The extracellular signal-related kinases (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets1. Here we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs 2-7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf reduced to ~30% of normal exhibit postnatal multisuture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to distal regulatory elements containing RUNX or AP1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes. PMID:23354439

  11. 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.

  12. Role of adenosine A2A receptor in cerebral ischemia reperfusion injury: Signaling to phosphorylated extracellular signal-regulated protein kinase (pERK1/2).

    PubMed

    Mohamed, R A; Agha, A M; Abdel-Rahman, A A; Nassar, N N

    2016-02-01

    Following brain ischemia reperfusion (IR), the dramatic increase in adenosine activates A2AR to induce further neuronal damage. Noteworthy, A2A antagonists have proven efficacious in halting IR injury, however, the detailed downstream signaling remains elusive. To this end, the present study aimed to investigate the possible involvement of phospho-extracellular signal-regulated kinase (pERK1/2) pathway in mediating protection afforded by the central A2A blockade. Male Wistar rats (250-270 g) subjected to bilateral carotid occlusion for 45 min followed by a 24-h reperfusion period showed increased infarct size corroborating histopathological damage, memory impairment and motor incoordination as well as increased locomotor activity. Those events were mitigated by the unilateral intrahippocampal administration of the selective A2A antagonist SCH58261 via a decrease in pERK1/2 downstream from diacyl glycerol (DAG) signaling. Consequent to pERK1/2 inhibition, reduced hippocampal microglial activation, glial tumor necrosis factor-alpha (TNF-α) and brain-derived neurotropic factor (BDNF) expression, glutamate (Glu), inducible nitric oxide synthase (iNOS) and thiobarbituric acid reactive substances (TBARS) were evident in animals receiving SCH58261. Additionally, the anti-inflammatory cytokine interleukin-10 (IL-10) increased following nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). Taken all together, these events suppressed apoptotic pathways via a reduction in cytochrome c (Cyt. c) as well as caspase-3 supporting a crucial role for pERK1/2 inhibition in consequent reduction of inflammatory and excitotoxic cascades as well as correction of the redox imbalance. PMID:26642806

  13. 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

  14. 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

  15. Evidence for Elevated Cerebrospinal Fluid ERK1/2 Levels in Alzheimer Dementia

    PubMed Central

    Spitzer, Philipp; Schieb, Heinke; Kamrowski-Kruck, Heike; Otto, Markus; Chiasserini, Davide; Parnetti, Lucilla; Herukka, Sanna-Kaisa; Schuchhardt, Johannes; Wiltfang, Jens; Klafki, Hans-Wolfgang

    2011-01-01

    Cerebrospinal fluid (CSF) samples from 33 patients with Alzheimer dementia (AD), 21 patients with mild cognitive impairment who converted to AD during followup (MCI-AD), 25 patients with stable mild cognitive impairment (MCI-stable), and 16 nondemented subjects (ND) were analyzed with a chemiluminescence immunoassay to assess the levels of the mitogen-activated protein kinase ERK1/2 (extracellular signal-regulated kinase 1/2). The results were evaluated in relation to total Tau (tTau), phosphorylated Tau (pTau), and beta-amyloid 42 peptide (Aβ42). CSF-ERK1/2 was significantly increased in the AD group as compared to stable MCI patients and the ND group. Western blot analysis of a pooled cerebrospinal fluid sample revealed that both isoforms, ERK1 and ERK2, and low amounts of doubly phosphorylated ERK2 were detectable. As a predictive diagnostic AD biomarker, CSF-ERK1/2 was inferior to tTau, pTau, and Aβ42. PMID:22145083

  16. ERK1/2 regulation of CD44 modulates oral cancer aggressiveness

    PubMed Central

    Judd, Nancy P.; Winkler, Ashley E.; Murillo-Sauca, Oihana; Brotman, Joshua J.; Law, Jonathan H.; Lewis, James S.; Dunn, Gavin P.; Bui, Jack D.; Sunwoo, John B.; Uppaluri, Ravindra

    2011-01-01

    Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) incurs significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, we generated cell line models from DMBA-induced murine primary OSCC capable of tumor formation upon transplantation into immunocompetent wild-type mice. While several lines grew rapidly and were capable of metastasis, some grew slowly and did not metastasize. Aggressively growing lines displayed ERK1/2 activation, which stimulated expression of CD44, a marker associated with EMT and putative cancer stem cells. MEK inhibition upstream of ERK1/2 decreased CD44 expression and promoter activity and reduced cell migration and invasion. Conversely, MEK1 activation enhanced CD44 expression and promoter activity, whereas CD44 attenuation reduced in vitro migration and in vivo tumor formation. Extending these findings to freshly resected human OSCC, we confirmed a strict relationship between ERK1/2 phosphorylation and CD44 expression. In summary, our findings identify CD44 as a critical target of ERK1/2 in promoting tumor aggressiveness and offer a preclinical proof of concept to target this pathway as a strategy to treat head and neck cancer. PMID:22086849

  17. 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 ...

  18. A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics

    PubMed Central

    Chaikuad, Apirat; Tacconi, Eliana; Zimmer, Jutta; Liang, Yanke; Gray, Nathanael S.; Tarsounas, Madalena; Knapp, Stefan

    2014-01-01

    Activation of the ERK pathway is a hallmark of cancer and targeting of upstream signalling partners led to the development of approved drugs. Recently SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induced a so far unknown binding pocket that accommodated the piperazine-phenyl-pyrimidine decoration. This novel binding pocket was created by an inactive conformation of the phosphate binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed canonical but two distinct type-I binding modes. Intriguingly, the novel binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity. PMID:25195011

  19. Regulation of Alveolar Epithelial Na+ Channels by ERK1/2 in Chlorine-Breathing Mice

    PubMed Central

    Lazrak, Ahmed; Chen, Lan; Jurkuvenaite, Asta; Doran, Stephen F.; Liu, Gang; Li, Qian; Lancaster, Jack R.

    2012-01-01

    The mechanisms by which the exposure of mice to Cl2 decreases vectorial Na+ transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na+ channels (ENaCs) after exposure to Cl2, and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (Iamil) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na+ channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl2, the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl2 increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased Iamil and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl2 decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in Iamil and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl2 activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity. PMID:21997487

  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. 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

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

    PubMed Central

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

    2013-01-01

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

  3. 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.

  4. 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.

  5. IKVAV regulates ERK1/2 and Akt signalling pathways in BMMSC population growth and proliferation

    PubMed Central

    Li, B; Qiu, T; Zhang, P; Wang, X; Yin, Y; Li, S

    2014-01-01

    Objectives The molecular mechanism of bone marrow mesenchymal stem cell (BMMSC) population growth and proliferation, induced by Isoleucyl-lysyl-valyl-alanyl-valine (IKVAV), was explored in this study. Materials and methods IKVAV peptides were synthesized by the solid-phase method. Influence of IKVAV on BMMSC population growth and proliferation were investigated by assays of CCK-8, flow cytometry, real-time PCR and western blotting. Results IKVAV peptide was found to induce proliferation and proliferating cell nuclear antigen (PCNA) synthesis of BMMSC in a dose- and time-dependent manner. Cell cycle analysis showed that the proportion of IKVAV-treated BMMSC in S phase in was higher than controls. Western blot results suggested that mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathways were activated by IKVAV by enhancing phosphorylation levels of ERK1/2 and Akt in the BMMSCs. Meanwhile, phosphorylation levels of ERK1/2 and Akt were partially blocked by ERK1/2 inhibitor (PD98059) and Akt inhibitor (wortmannin), respectively. Conclusions Our results demonstrated that IKVAV stimulated BMMSC population growth and proliferation by activating MAPK/ERK1/2 and PI3K/Akt signalling pathways. This study is the first to reveal an enhancement effect of IKVAV peptide on BMMSC at the signal transduction level, and the outcome could provide experimental evidence for application of IKVAV-grafted scaffolds in the field of BMMSC-based tissue engineering. PMID:24617901

  6. 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.

  7. 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

  8. Knockout of ERK1 enhances cocaine-evoked immediate early gene expression and behavioral plasticity.

    PubMed

    Ferguson, Susan M; Fasano, Stefania; Yang, Pengwei; Brambilla, Riccardo; Robinson, Terry E

    2006-12-01

    The ability of cocaine to produce lasting neural adaptations in mesocorticolimbic brain regions is thought to promote drug seeking and facilitate addiction in humans. The Ras-controlled Raf-MEK-ERK protein kinase signaling cascade has been implicated in the behavioral and neurobiological actions of cocaine in animals. However, these pharmacological studies have not been able to determine the specific role of the two predominant isoforms of ERK (ERK1 and ERK2) in these processes. We report here that deletion of the ERK1 isoform, which leads to increased ERK2 stimulus-dependent signaling, facilitates the development of cocaine-induced psychomotor sensitization and the acquisition of a cocaine conditioned place preference. Conversely, pharmacological blockade of ERK signaling attenuates the development of psychomotor sensitization to cocaine. Finally, cocaine-evoked gene expression in mesocorticolimbic brain regions is potentiated in ERK1-deficient mice. Thus, alterations in ERK signaling influence both the neurobiological impact of cocaine and its ability to produce enduring forms of drug experience-dependent behavioral plasticity. Our results suggest that enhanced ERK2 signaling following repeated drug exposure may facilitate the development of forms of cocaine-induced plasticity that contribute to addiction. PMID:16407894

  9. 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

  10. 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.

  11. Concomitant activation of the PI3K/Akt and ERK1/2 signalling is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells.

    PubMed

    Yin, Jian; Hao, Zhichao; Ma, Yuanyuan; Liao, Shuang; Li, Xianxian; Fu, Jing; Wu, Yeke; Shen, Jiefei; Zhang, Ping; Li, Xiaoyu; Wang, Hang

    2014-05-01

    IL-6 has a dual role in bone remodelling. The ERK1/2 pathway partially upregulated IL-6 secretion in osteocyte-like MLO-Y4 cells exposed to CCF. We have now investigated the possible role of phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway in the CCF-induced IL-6 expression. MLO-Y4 cells were treated with CCF 2,000 µstrain, 2 Hz, or 10, 30 min, 1, 3 and 6 h. IL-6 expression, Akt and ERK1/2 and PI3K/Akt phosphorylation were determined by RT-PCR, ELISA and Western blotting. Inhibition of PI3K/Akt with LY294002 or ERK1/2 with PD98059 significantly attenuated IL-6 upregulation, and IL-6 expression was abolished by inhibiting both pathways. Inhibition of one pathway downregulated the other's phosphorylation level. In conclusion, concomitant activation of PI3K/Akt and ERK1/2 pathways mediated IL-6 expression in MLO-Y4 cells under CCF. PMID:24375569

  12. Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

    PubMed Central

    Zhan, Qiong; Song, Rui; Zeng, Qingchun; Yao, Qingzhou; Ao, Lihua; Xu, Dingli; Fullerton, David A.; Meng, Xianzhong

    2015-01-01

    Calcific aortic valve disease (CAVD) is characterized by chronic inflammation and progressive calcification in valve leaflets. Aortic valve interstitial cells (AVICs) play a critical role in the pathogenesis of CAVD. Previous studies show that stimulation of Toll-like receptor (TLR) 2 or TLR4 in AVICs in vitro up-regulates the expression of osteogenic mediators. Double-stranded RNA (dsRNA) can activate pro-inflammatory signaling through TLR3, the NLRP3 inflammasome and RIG-I-like receptors. The objective of this study is to determine the effect of dsRNA on AVIC osteogenic activities and the mechanism of its action. Methods and results: AVICs isolated from normal human valves were exposed to polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Treatment with poly(I:C) increased the production of bone morphogenetic protein-2 (BMP-2), transforming growth factor beta-1 (TGF-β1) and alkaline phosphatase (ALP), and resulted in calcium deposit formation. Poly(I:C) induced the phosphorylation of NF-κB and ERK1/2. Knockdown of TLR3 essentially abrogated NF-κB and ERK1/2 phosphorylation, and markedly reduced the effect of poly(I:C) on the production of BMP-2, TGF-β1 and ALP. Further, inhibition of either NF-κB or ERK1/2 markedly reduced the levels of BMP-2, TGF-β1 and ALP in cells exposed to poly(I:C). Conclusion: Poly(I:C) up-regulates the production of BMP-2, TGF-β1 and ALP, and promotes calcium deposit formation in human AVICs. The pro-osteogenic effect of poly(I:C) is mediated primarily by TLR3 and the NF-κB and ERK1/2 pathways. These findings suggest that dsRNA, when present in aortic valve tissue, may promote CAVD progression through up-regulation of AVIC osteogenic activities. PMID:25798067

  13. TGF-β1 antagonizes TNF-α induced up-regulation of matrix metalloproteinase 3 in nucleus pulposus cells: role of the ERK1/2 pathway.

    PubMed

    Yang, Hao; Gao, Fei; Li, Xiang; Wang, Jianru; Liu, Hui; Zheng, Zhaomin

    2015-11-01

    Tumor necrosis factor-α (TNF-α) has been shown to have a catabolic effect on intervertebral disc degeneration (IVDD), including increasing MMP3 expression and subsequent extracellular matrix (ECM) degradation. In contrast, transforming growth factor-β1 (TGF-β1) has an anabolic effect on nucleus pulposus (NP) cells. However, the anti-catabolic effect of TGF-β1 under inflammatory condition is unknown. The aim of this study was to demonstrate whether TGF-β1 can reverse TNF-α-induced MMP3 increase in NP cells and to further investigate the underlying mechanisms. The transcriptional activity, gene expression, and protein levels of MMP3 were measured by luciferase reporter assay, qRT-PCR and western blot, respectively. TNF-α increased MMP3 expression in rat NP cells time and dose dependently. TGF-β1 could abolish TNF-α-mediated up-regulation of collagen I and MMP3 expression, and down-regulate aggrecan and collagen II expression. The ERK1/2 signaling pathway was activated after exposure to TGF-β1. Treatment with ERK1/2 inhibitors (PD98059 and U0126) abolished the antagonistic effect of TGF-β1 on TNF-α mediated catabolic responses. These findings provide novel evidence supporting the anti-catabolic role of TGF-β1 in IVDD, which is important for the potential clinical application of TGF-β1 in disc degenerative disorders. PMID:26075533

  14. 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.

  15. Clematichinenoside Serves as a Neuroprotective Agent Against Ischemic Stroke: The Synergistic Action of ERK1/2 and cPKC Pathways

    PubMed Central

    Liu, Chao; Du, Qianming; Zhang, Xu; Tang, Zhichao; Ji, Hui; Li, Yunman

    2016-01-01

    There are numerous evidences suggesting that inhibition of apoptosis of neurons play a critical role in preventing the damage and even death of neurons after brain ischemia/reperfusion, which shows therapeutic potential for clinical treatment of brain injury induced by stroke. In this study, we aimed to investigate the neuroprotective effect of Clematichinenoside (AR) and its underlying mechanisms. MCAO mode was performed in rats and OGD/R model in primary cortical neurons to investigate the neuroprotective effect of AR. The rate of apoptotic cells was measured using TUNEL assay in cerebral cortex and flow cytometric assay in cortical neurons. Apoptosis-related proteins such as bcl-2, bcl-xl, and bax and the phosphorylation of ERK1/2, cPKC, p90RSK, and CREB in ischemic penumbra were assayed by western blot. Furthermore, we made a thorough inquiry about how these proteins play roles in the anti-apoptotic mechanism using targets-associated inhibitors step by step. The results revealed that AR could activate both ERK1/2 and cPKC which resulted in p90RSK phosphorylation and translocation into the nucleus. Moreover, CREB, a downstream target of p90RSK, was phosphorylated and then bound to cAMP-regulated enhancer (CRE) to activate apoptosis-related genes, and finally ameliorate ischemic stroke through preventing neuron death. In conclusion, these data strongly suggest that AR could be used as an effective neuroprotective agent to protect against ischemic stroke after cerebral I/R injury through regulating both ERK1/2 and cPKC mediated p90RSK/CREB apoptotic pathways. PMID:26793066

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

    PubMed Central

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

    2015-01-01

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

  17. Pathogenesis of Lyme neuroborreliosis: mitogen-activated protein kinases Erk1, Erk2, and p38 in the response of astrocytes to Borrelia burgdorferi lipoproteins.

    PubMed

    Ramesh, Geeta; Philipp, Mario T

    Lyme borreliosis, which is prevalent both in the US and in Europe, is an infectious disease that may cause local inflammation in numerous organs. We have hypothesized that, as with some neurodegenerative diseases, the pathogenesis of the neurocognitive deficiencies associated with Lyme neuroborreliosis of the central nervous system also has an inflammatory component. Dysregulated production of pro-inflammatory cytokines such as IL-6 and TNF-alpha can lead to neuronal damage. Mitogen-activated protein kinases (MAPK) play a key role in the regulation of neuronal development, growth, and survival, as well as that of pro-inflammatory cytokine production. As a model, we explored the possibility that MAPK-mediated lipoprotein-induced apoptosis and gliosis of rhesus monkey astrocytes stimulated in vitro. Lipoproteins are the key inflammatory molecule type of Borrelia burgdorferi, the spirochete that causes Lyme disease, and we had previously shown that lipoprotein-induced TNF-alpha production in astrocytes caused astrocyte apoptosis, and IL-6 enhanced proliferation of these cells. Lipoproteins readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that spirochetes may address as they invade the central nervous system. We also investigated whether specific inhibition of p38 and Erk1/2 MAPK would inhibit TNF-alpha and IL-6 production and thus astrocyte apoptosis, and proliferation, respectively. Lipoprotein-stimulated IL-6 production was unaffected by the MAPK inhibitors. In contrast, inhibition of both p38 and Erk1/2 significantly diminished TNF-alpha production, and totally abrogated production of this cytokine when both MAPK pathways were inhibited simultaneously. MAPK inhibition thus may be considered as a strategy to control inflammation and apoptosis in Lyme neuroborreliosis. PMID:15893422

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

    PubMed

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

    2015-04-15

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

  19. 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.

  20. Antagonistic Interaction between Systemic Acquired Resistance and the Abscisic Acid–Mediated Abiotic Stress Response in Arabidopsis[W

    PubMed Central

    Yasuda, Michiko; Ishikawa, Atsushi; Jikumaru, Yusuke; Seki, Motoaki; Umezawa, Taishi; Asami, Tadao; Maruyama-Nakashita, Akiko; Kudo, Toshiaki; Shinozaki, Kazuo; Yoshida, Shigeo; Nakashita, Hideo

    2008-01-01

    Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plants, such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana, is mediated by salicylic acid (SA). Here, using two types of SAR-inducing chemicals, 1,2-benzisothiazol-3(2H)-one1,1-dioxide and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, which act upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. In an analysis using several mutants in combination with these chemicals, treatment with ABA suppressed SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene-mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to be ABA dependent. Conversely, the activation of SAR suppressed the expression of ABA biosynthesis–related and ABA-responsive genes, in which the NPR1 protein or signaling downstream of NPR1 appears to contribute. Therefore, our data have revealed that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses. PMID:18586869

  1. Acute morphine affects the rat circadian clock via rhythms of phosphorylated ERK1/2 and GSK3β kinases and Per1 expression in the rat suprachiasmatic nucleus

    PubMed Central

    Pačesová, Dominika; Volfová, Barbora; Červená, Kateřina; Hejnová, Lucie; Novotný, Jiří; Bendová, Zdeňka

    2015-01-01

    Background and Purpose Opioids affect the circadian clock and may change the timing of many physiological processes. This study was undertaken to investigate the daily changes in sensitivity of the circadian pacemaker to an analgesic dose of morphine, and to uncover a possible interplay between circadian and opioid signalling. Experimental Approach A time-dependent effect of morphine (1 mg·kg−1, i.p.) applied either during the day or during the early night was followed, and the levels of phosphorylated ERK1/2, GSK3β, c-Fos and Per genes were assessed by immunohistochemistry and in situ hybridization. The effect of morphine pretreatment on light-induced pERK and c-Fos was examined, and day/night difference in activity of opioid receptors was evaluated by [35S]-GTPγS binding assay. Key Results Morphine stimulated a rise in pERK1/2 and pGSK3β levels in the suprachiasmatic nucleus (SCN) when applied during the day but significantly reduced both kinases when applied during the night. Morphine at night transiently induced Period1 but not Period2 in the SCN and did not attenuate the light-induced level of pERK1/2 and c-Fos in the SCN. The activity of all three principal opioid receptors was high during the day but decreased significantly at night, except for the δ receptor. Finally, we demonstrated daily profiles of pERK1/2 and pGSK3β levels in the rat ventrolateral and dorsomedial SCN. Conclusions and Implications Our data suggest that the phase-shifting effect of opioids may be mediated via post-translational modification of clock proteins by means of activated ERK1/2 and GSK3β. PMID:25828914

  2. Mycobacterium tuberculosis Mce3E suppresses host innate immune responses by targeting ERK1/2 signaling.

    PubMed

    Li, Jie; Chai, Qi-Yao; Zhang, Yong; Li, Bing-Xi; Wang, Jing; Qiu, Xiao-Bo; Liu, Cui Hua

    2015-04-15

    Crucial to the pathogenesis of the tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis is its ability to subvert host immune defenses to promote its intracellular survival. The mammalian cell entry protein 3E (Mce3E), located in the region of difference 15 of the M. tuberculosis genome and absent in Mycobacterium bovis bacillus Calmette-Guérin, has an essential role in facilitating the internalization of mammalian cells by mycobacteria. However, relatively little is known about the role of Mce3E in modulation of host innate immune responses. In this study, we demonstrate that Mce3E inhibits the activation of the ERK1/2 signaling pathway, leading to the suppression of Tnf and Il6 expression, and the promotion of mycobacterial survival within macrophages. Mce3E interacts and colocalizes with ERK1/2 at the endoplasmic reticulum in a DEF motif (an ERK-docking motif)-dependent manner, relocates ERK1/2 from cytoplasm to the endoplasmic reticulum, and finally reduces the association of ERK1/2 with MEK1 and blocks the nuclear translocation of phospho-ERK1/2. A DEF motif mutant form of Mce3E (F294A) loses its ability to suppress Tnf and Il6 expression and to promote intracellular survival of mycobacteria. Inhibition of the ERK1/2 pathway in macrophages using U0126, a specific inhibitor of the ERK pathway, also leads to the suppressed Tnf and Il6 expression and the enhanced intracellular survival of mycobacteria. Taken together, these results suggest that M. tuberculosis Mce3E exploits the ERK1/2 signaling pathway to suppress host innate immune responses, providing a potential Mce3E-ERK1/2 interface-based drug target against M. tuberculosis. PMID:25780035

  3. 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.

  4. 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

  5. NGF increases VEGF expression and promotes cell proliferation via ERK1/2 and AKT signaling in Müller cells

    PubMed Central

    Wang, Jing; He, Chang; Zhou, Tian; Huang, Zijing; Zhou, Lingli

    2016-01-01

    Purpose Nerve growth factor (NGF) is a classic neuroprotective factor that contributes to angiogenesis under pathological conditions, which might be mediated by the upregulation of vascular endothelial growth factor (VEGF). Retinal Müller cells are a critical source of growth factors, including NGF and VEGF, and express the receptor for NGF, indicating the functional significance of NGF signaling in Müller cells. The aim of this study is to explore the effect of NGF on the production of other growth factors and cellular proliferation in Müller cells and to further detect the potential mechanism of these effects. Methods Primary Müller cells from C57BL/6J mice were isolated and identified with glutamine synthetase (GS) immunofluorescence (IF), a specific marker for Müller cells. TrkA, a high affinity receptor for NGF, was detected with IF staining in the primary Müller cells. Then, the cultured cells were stimulated with recombinant mouse NGF, and the supernatants and the cellular lysate were collected at different time points. VEGF secretion in the supernatant was detected with an enzyme-linked immunosorbent assay (ELISA). The signaling activation in the Müller cells was accessed by western blot using specific phosphorylated antibodies. In addition, cell proliferation was analyzed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Furthermore, K252a, U0126, and LY294002, the inhibitors for TrkA, extracellular signal-regulated kinases 1/2 (ERK1/2), and phosphatidylinositol 3-kinase (PI3K)/AKT, respectively, were used in combination with NGF in the assays analyzing VEGF expression and cell proliferation. Results Primary mouse Müller cells were successfully cultured and confirmed with GS positive staining. The IF results showed that the TrkA receptor was abundantly expressed on Müller cells. The ELISA results revealed that NGF significantly promoted the production and secretion of VEGF in Müller cells after 12 or 24 h of

  6. 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

  7. An acquired defect in IgG-dependent phagocytosis explains the impairment in antibody-mediated cellular depletion in Lupus.

    PubMed

    Ahuja, Anupama; Teichmann, Lino L; Wang, Haowei; Dunn, Robert; Kehry, Marilyn R; Shlomchik, Mark J

    2011-10-01

    B cells play important roles in autoimmune diseases ranging from multiple sclerosis to rheumatoid arthritis. B cells have also long been considered central players in systemic lupus erythematosus. However, anti-CD20-mediated B cell depletion was not effective in two clinical lupus studies, whereas anti-B lymphocyte stimulator, which inhibits B cell survival, was effective. Others and we previously found that anti-CD20-based depletion was surprisingly ineffective in tissues of lupus-prone mice, but that persistent high doses eventually led to depletion and ameliorated lupus. Lupus patients might also have incomplete depletion, as suggested in several studies, and which could have led to therapeutic failure. In this study, we investigated the mechanism of resistance to Ab-mediated cellular depletion in murine lupus. B cells from lupus-prone mice were easily depleted when transferred into normal environments or in lupus-prone mice that lacked serum Ig. Serum from lupus-prone mice transferred depletion resistance, with the active component being IgG. Because depletion is FcγR-dependent, we assayed macrophages and neutrophils exposed to lupus mouse serum, showing that they are impaired in IgG-mediated phagocytosis. We conclude that depletion resistance is an acquired, reversible phagocytic defect depending on exposure to lupus serum IgG. These results have implications for optimizing and monitoring cellular depletion therapy. PMID:21873531

  8. 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

  9. Activation of extracellular signal-regulated kinases Erk-1 and Erk-2 by cell swelling in H4IIE hepatoma cells.

    PubMed Central

    Schliess, F; Schreiber, R; Häussinger, D

    1995-01-01

    Hepatic metabolism and gene expression are among the factors controlled by the cellular hydration state, which changes within minutes in response to aniso-osmotic environments, cumulative substrate uptake, oxidative stress and under the influence of hormones such as insulin. The signalling events coupling cell-volume changes to altered cell function were studied in H4IIE rat hepatoma cells. Hypo-osmotic cell swelling resulted within 1 min in a tyrosine kinase-mediated activation of the extracellular signal-regulated protein kinases Erk-1 and Erk-2, which was independent of protein kinase C and cytosolic calcium. Activation of mitogen-activated protein kinases was followed by an increased phosphorylation of c-Jun, which may explain our recently reported finding of an about 5-fold increase in c-jun mRNA level in response to cell swelling. Pretreatment of cells with pertussis or cholera toxin abolished the swelling-induced activation of Erk-1 and Erk-2, suggesting the involvement of G-proteins. Thus, a signal-transduction pathway resembling growth factor signalling is activated already by osmotic water shifts across the plasma membrane, thereby providing a new perspective for adaption of cell function to alterations of the environment. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7619047

  10. Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis

    PubMed Central

    Feng, Jian Q.; Guo, Feng-Jin; Jiang, Bai-Chun; Zhang, Yan; Frenkel, Sally; Wang, Da-Wei; Tang, Wei; Xie, Yixia; Liu, Chuan-Ju

    2010-01-01

    Granulin epithelin precursor (GEP) has been implicated in development, tissue regeneration, tumorigenesis, and inflammation. Herein we report that GEP stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo, and GEP-knockdown mice display skeleton defects. Similar to bone morphogenic protein (BMP) 2, application of the recombinant GEP accelerates rabbit cartilage repair in vivo. GEP is a key downstream molecule of BMP2, and it is required for BMP2-mediated chondrocyte differentiation. We also show that GEP activates chondrocyte differentiation through Erk1/2 signaling and that JunB transcription factor is one of key downstream molecules of GEP in chondrocyte differentiation. Collectively, these findings reveal a novel critical role of GEP growth factor in chondrocyte differentiation and the molecular events both in vivo and in vitro.—Feng, J. Q., Guo, F.-J., Jiang, B.-C., Zhang, Y., Frenkel, S., Wang, D.-W., Tang, W., Xie, Y., Liu, C.-J. Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis. PMID:20124436

  11. MK615, a Prunus mume Steb. Et Zucc ('Ume') extract, attenuates the growth of A375 melanoma cells by inhibiting the ERK1/2-Id-1 pathway.

    PubMed

    Tada, Ko-ichi; Kawahara, Ko-ichi; Matsushita, Shigeto; Hashiguchi, Teruto; Maruyama, Ikuro; Kanekura, Takuro

    2012-06-01

    The Japanese apricot, a commonly consumed food called 'Ume' in Japan, has been used for a traditional Japanese medicine for centuries. MK615, an extract of compounds from 'Ume', has strong antitumorigenic and antiinflammatory effects including the induction of apoptosis and autophagy, and inhibition of cytokine production mediated via the inhibition of MAPKs signaling including ERK-1/2, JNK and p38MAPK. The inhibitor of DNA binding 1 (Id-1), a basic helix-loop-helix (bHLH) transcription factor family, is essential for DNA binding and the transcriptional regulation of various proteins that play important roles in the development, progression and invasion of tumors. In melanoma, Id-1 is constitutively expressed in the late and early stages, suggesting it as a therapeutic target in patients with melanoma. This study reports that MK615 profoundly reduced both the mRNA- and protein expression levels of Id-1 and inhibited cell growth in A375 melanoma cells. MK615 markedly inhibited the phosphorylation of ERK1/2, which is associated with Id-1 protein expression in A375 cells. Id-1-specific RNAi induced the death of A375 cells. Moreover, the expression of Bcl-2 was decreased by both MK615 and Id-1-specific RNAi in A375 cells. The results suggest that MK615 is a potential therapeutic agent for treating malignant melanoma. PMID:22076920

  12. 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.

  13. Cardiac inflammation involving in PKCε or ERK1/2-activated NF-κB signalling pathway in mice following exposure to titanium dioxide nanoparticles.

    PubMed

    Yu, Xiaohong; Hong, Fashui; Zhang, Yu-Qing

    2016-08-01

    The evaluation of toxicological effects of nanoparticles (NPs) is increasingly important due to their growing occupational use and presence as compounds in consumer products. Recent researches have demonstrated that long-term exposure to air particulate matter can induce cardiovascular events, but whether cardiovascular disease, such as cardiac damage, is induced by NP exposure and its toxic mechanisms is rarely evaluated. In the present study, when mice were continuously exposed to TiO2 NPs at 2.5, 5 or 10mg/kg BW by intragastric administration for 90days, obvious histopathological changes, and great alterations of NF-κB and its inhibitor I-κB, as well as TNF-α, IL-1β, IL-6 and IFN-α expression were induced. The NPs significantly decreased Ca(2+)-ATPase, Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase activities and enhanced NCX-1 content. The NPs also considerably increased CAMK II and α1/β1-AR expression and up-regulated p-PKCε and p-ERK1/2 in a dose-dependent manner in the mouse heart. These data suggest that low-dose and long-term exposure to TiO2 NPs may cause cardiac damage such as cardiac fragmentation or disordered myocardial fibre arrangement, tissue necrosis, myocardial haemorrhage, swelling or cardiomyocyte hypertrophy, and the inflammatory response was potentially mediated by NF-κB activation via the PKCε or ERK1/2 signalling cascades in mice. PMID:27054666

  14. Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment.

    PubMed

    Zippelius, Alfred; Schreiner, Jens; Herzig, Petra; Müller, Philipp

    2015-03-01

    CD40 stimulation on antigen-presenting cells (APC) allows direct activation of CD8(+) cytotoxic T cells, independent of CD4⁺ T-cell help. Agonistic anti-CD40 antibodies have been demonstrated to induce beneficial antitumor T-cell responses in mouse models of cancer and early clinical trials. We report here that anti-CD40 treatment induces programmed death ligand-1 (PD-L1) upregulation on tumor-infiltrating monocytes and macrophages, which was strictly dependent on T cells and IFNγ. PD-L1 expression could be counteracted by coadministration of antibodies blocking the PD-1 (programmed death-1)/PD-L1 axis as shown for T cells from tumor models and human donors. The combined treatment was highly synergistic and induced complete tumor rejection in about 50% of mice bearing MC-38 colon and EMT-6 breast tumors. Mechanistically, this was reflected by a strong increase of IFNγ and granzyme-B production in intratumoral CD8⁺ T cells. Concomitant CTLA-4 blockade further improved rejection of established tumors in mice. This study uncovers a novel mechanism of acquired resistance upon agonistic CD40 stimulation and proposes that the concomitant blockade of the PD-1/PD-L1 axis is a viable therapeutic strategy to optimize clinical outcomes. PMID:25623164

  15. 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

  16. Two novel ALK mutations mediate acquired resistance to the next generation ALK inhibitor alectinib

    PubMed Central

    Katayama, Ryohei; Friboulet, Luc; Koike, Sumie; Lockerman, Elizabeth L.; Khan, Tahsin M.; Gainor, Justin F.; Iafrate, A. John; Takeuchi, Kengo; Taiji, Makoto; Okuno, Yasushi; Fujita, Naoya; Engelman, Jeffrey A.; Shaw, Alice T.

    2014-01-01

    Purpose The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. Experimental Design We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity-relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. Results We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. Conclusions We have identified two novel ALK mutations arising after alectinib exposure which are sensitive to other next generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. PMID:25228534

  17. [IL-33 promotes airway remodeling in a mouse model of asthma via ERK1/2 signaling pathway].

    PubMed

    Zhang, Yuanyuan; Bian, Cuixia; Wu, Jinxiang; Zhao, Jiping; Wang, Junfei; Liu, Tian; Liu, Lin; Dong, Liang

    2016-05-01

    Objective To explore the role of IL-33 in asthmatic airway remodeling. Methods Male BALB/c mice were randomly divided into 3 groups: a control group, an ovalbumin (OVA) group, and an anti-IL-33 antibody combined with OVA group. The airway remodeling features in mice were observed by HE staining. In addition, the expressions of IL-33, alpha smooth muscle actin (α-SMA), and type 1 collagen (Col1) in the airway of mice were detected by immunohistochemistry and Western blotting. Finally, Western blotting was used to determine the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen- and stress-activated protein kinase 1 (MSK1) in the lungs of mice. In vitro, human lung fibroblasts (HLF-1) were pretreated with the ERK1/2 inhibitor U0126 or the MSK1 inhibitor H89 respectively, and then treated with the human recombinant IL-33 (rIL-33). Then real-time quantitative PCR and Western blotting were used to test the expressions of α-SMA and Col1. Immunofluorescence cytochemistry and Western blotting were also used to observe the phosphorylation of ERK1/2 and MSK1 in HLF-1 cells. Results The pre-treatment with the ERK1/2 inhibitor U0126 or anti-IL-33 antibody significantly abolished the OVA-induced airway remodeling, increased expressions of IL-33, α-SMA, Col1, and phosphorylation of ERK1/2 and MSK1 in the airway of mice. In vitro, the increased expressions of α-SMA and Col1 and the phosphorylation of ERK1/2 and MSK1 induced by rIL-33 in HLF-1 cells were markedly inhibited by the pre-treatment with U0126 or H89. Conclusion IL-33 promotes airway remodeling in asthmatic mice via the ERK1/2-MSK1 signaling pathway. PMID:27126934

  18. Varicella-Zoster Virus ORF12 Protein Triggers Phosphorylation of ERK1/2 and Inhibits Apoptosis

    PubMed Central

    Liu, XueQiao; Li, Qingxue; Dowdell, Kennichi; Fischer, Elizabeth R.

    2012-01-01

    Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases involved in many cellular processes, including cell proliferation, differentiation, inflammation, and cell death. Activation of several MAPKs, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), results in stimulation of activator protein 1 (AP-1), which promotes gene transcription. Previous studies have demonstrated that varicella-zoster virus (VZV) infection activates ERK1/2, p38, and JNK to promote viral replication, but the underlying mechanism(s) is unclear. To identify viral proteins responsible for the activation of MAPK, we used a proteomic approach to screen viral proteins for AP-1 promoter activation by an AP-1–luciferase reporter assay. We found that VZV ORF12 protein, located in the tegument of virions, enhances AP-1 reporter activity. This effect of ORF12 protein was markedly inhibited by a MAPK/ERK kinase 1 and 2 (MEK1/2) inhibitor (U0126), partially blocked by a p38 inhibitor (SB202190), but not inhibited by a JNK inhibitor (SP600125). Expression of VZV ORF12 protein in cells resulted in phosphorylation of ERK1/2 and p38 but not JNK. Infection of cells with a VZV ORF12 deletion mutant resulted in reduced levels of phosphorylated ERK1/2 (p-ERK1/2) compared to infection with wild-type VZV. Furthermore, deletion of ORF12 rendered VZV-infected cells more susceptible to staurosporine-induced apoptosis. In conclusion, VZV ORF12 protein activates the AP-1 pathway by selectively triggering the phosphorylation of ERK1/2 and p38. Cells infected with a VZV ORF12 deletion mutant have reduced levels of p-ERK1/2 and are more susceptible to apoptosis than cells infected with wild-type VZV. PMID:22238304

  19. 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.

  20. Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1

    PubMed Central

    Lai, Shenshen; Pelech, Steven

    2016-01-01

    The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Their phosphorylation within the well-known activation T-loop, a variable region between protein kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. Extracellular signal–regulated kinase 1 (ERK1), a member of the extensively studied mitogen-activated protein kinase (MAPK) family, serves as a paradigm for regulation of protein kinases in signaling modules. In addition to the well-documented T202 and Y204 stimulatory phosphorylation sites in the activation T-loop of ERK1 and its closest relative, ERK2, three additional flanking phosphosites have been confirmed (T198, T207, and Y210 from ERK1) by high-throughput mass spectrometry. In vitro kinase assays revealed the functional importance of T207 and Y210, but not T198, in negatively regulating ERK1 catalytic activity. The Y210 site could be important for proper conformational arrangement of the active site, and a Y210F mutant could not be recognized by MEK1 for phosphorylation of T202 and Y204 in vitro. Autophosphorylation of T207 reduces the catalytic activity and stability of activated ERK1. We propose that after the activation of ERK1 by MEK1, subsequent slower phosphorylation of the flanking sites results in inhibition of the kinase. Because the T207 and Y210 phosphosites of ERK1 are highly conserved within the eukaryotic protein kinase family, hyperphosphorylation within the kinase activation T-loop may serve as a general mechanism for protein kinase down-regulation after initial activation by their upstream kinases. PMID:26823016

  1. Isoalantolactone Enhances the Radiosensitivity of UMSCC-10A Cells via Specific Inhibition of Erk1/2 Phosphorylation

    PubMed Central

    Hu, Jiehua; Wang, Hongyan; Li, Lihua; Liu, Hua

    2015-01-01

    Background Although radiotherapy is one of the mainstream approaches for the treatment of head and neck squamous cell carcinoma (HNSCC), this cancer is always associated with resistance to radiation. In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells. Methods The radiosensitization of UMSCC-10A cells treated with isoalantolactone was analyzed by colony formation assay. The radiosensitization effects of isoalantolactone on cell proliferation, cell cycle and apoptosis regulation were examined by BrdU incorporation assay, DNA content assay and flow cytometry, respectively. Western blotting was performed to determine the effects of isoalantolactone combined with radiation on the protein expression of Mek, extracellular signal-regulated kinase (Erk1/2) as well as phosphorylated Mek and Erk1/2. Erk1/2 knockdown by siRNA was used to confirm that isoalantolactone specifically inhibited the activation of Erk1/2 signaling pathway in UMSCC-10A cells. Results Isoalantolactone enhanced the radiosensitivity of UMSCC-10A cells; the sensitivity enhanced ratios (SERs) were 1.44 and 1.63, respectively, for 2.5 and 5 μM. Moreover, isoalantolactone enhanced radiation-induced cell proliferation and apoptosis and cell cycle arrested at G2/M phase. Furthermore, no marked changes were observed in the expression of total Erk1/2 and Mek protein after radiation treatment. However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent. In addition, the radiosensitivity of UMSCC-10A cells with Erk1/2 knockdown was increased. Isoalantolactone cannot further prevent the proliferation of UMSCC-10A cells with Erk1/2 knockdown which other mechanism regulated cell proliferation. Conclusion Our results suggested that isoalantolactone enhanced radiation-induced apoptosis, cell cycle arrested and reduced the cell

  2. 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

  3. 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.

  4. 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

    2016-01-01

    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

  5. Lutropin/choriogonadotropin (LH/CG) stimulate the proliferation of primary cultures of rat Leydig cells through a pathway that involves activation of the ERK1/2 cascade*

    PubMed Central

    Shiraishi, Koji; Ascoli, Mario

    2007-01-01

    Primary cultures of progenitor and immature rat Leydig cells were established from the testes of 21 and 35 day old rats, respectively. The cell population remained homogeneous after 4–6 days in culture as judged by staining for 3β-hydroxysteroid dehydrogenase but the cells were unable to bind 125I-hCG or to respond to hCG with classical LH receptor (LHR)-mediated responses including cAMP and inositol phosphate accumulation, steroid biosynthesis or the phosphorylation of the extracellular regulated kinases 1/2 (ERK1/2). Infection of primary cultures with recombinant adenovirus coding for β-galactosidase showed that ~65% of the cells are infected. Infection with adenovirus coding for the human LHR (hLHR) allowed for expression of the hLHR at a density of ~25,000 receptors/cell and allowed the cells to respond to hCG with increases in cAMP and inositol phosphate accumulation, steroid biosynthesis and the phosphorylation of ERK1/2. Although progenitor and immature cells were able to respond to hCG with an increase in progesterone, only the immature cells responded with an increase in testosterone. In addition to these classical LHR-mediated responses the primary cultures of progenitor or immature rat Leydig cells expressing the recombinant hLHR proliferated robustly when incubated with hCG and this proliferative response was sensitive to an inhibitor of ERK1/2 phosphorylation. These studies establish a novel experimental paradigm that can be used to study the proliferative response of Leydig cells to LH/CG. We conclude that activation of the LHR-provoked Leydig cell proliferation requires activation of the ERK1/2 cascade. PMID:17412805

  6. 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

  7. 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

  8. 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

  9. Transforming growth factor-β1 induces epithelial-to-mesenchymal transition in human lung cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways.

    PubMed

    Chen, Xiao-Feng; Zhang, Hui-Jun; Wang, Hai-Bing; Zhu, Jun; Zhou, Wen-Yong; Zhang, Hui; Zhao, Ming-Chuan; Su, Jin-Mei; Gao, Wen; Zhang, Lei; Fei, Ke; Zhang, Hong-Tao; Wang, He-Yong

    2012-04-01

    Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-β1 (TGF-β1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-β1 for 48 h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-β1-induced EMT after 48 h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-β1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-β1-induced EMT of A549 cells. PMID:21713404

  10. Markers of tyrosine kinase activity in eosinophilic esophagitis: A pilot study of the FIP1L1-PDGFRα fusion gene, pERK 1/2, and pSTAT5

    PubMed Central

    Dellon, Evan S.; Bower, Jacquelyn J.; Keku, Temitope O.; Chen, Xiaoxin; Miller, C. Ryan; Woosley, John T.; Orlando, Roy C.; Shaheen, Nicholas J.

    2011-01-01

    Background and Aims The pathogenesis of eosinophilic esophagitis (EoE) is incompletely understood. In certain eosinophilic diseases, activation of tyrosine kinase after fusion of the Fip1-like-1 and platelet-derived growth factor receptor-α genes (F-P fusion gene) mediates eosinophilia via downstream effectors such as extracellular-regulated kinase (ERK1/2) and signal transducers and activators of transcription (STAT5). This mechanism has not been examined in EoE. Our aim was to detect the F-P fusion gene, pERK1/2, and pSTAT5 in esophageal tissue from patients with EoE, gastroesophageal reflux disease (GERD), and normal controls. Materials and Methods We performed a cross-sectional pilot study comparing patients with steroid-responsive and steroid-refractory EoE, to GERD patients and normal controls. EoE cases were defined by consensus guidelines. Fluorescence in-situ hybridization (FISH) was performed to detect the F-P fusion gene and immunohistochemistry (IHC) was performed to detect pERK1/2 and pSTAT5 in esophageal biopsies. Results Twenty-nine subjects (median age 30 yrs (range 1–59); 16 male; 24 Caucasian) were included: 8 normal, 6 GERD, and 15 EoE (5 steroid-refractory). On FISH, 98%, 99%, and 99% of the nuclei in the normal, GERD, and EoE groups, respectively, were normal (p=0.42). On IHC, a median of 250, 277, and 479 nuclei/mm2 stained for pERK1/2 in the normal, GERD, and EoE groups, respectively (p=0.07); the refractory EoE patients had the highest degree pERK1/2 staining (846 nuclei/mm2; p=0.07). No trend was seen for pSTAT5. Conclusions The F-P fusion gene was not detected with increased frequency in EoE. Patients with EoE had a trend towards higher levels of pERK1/2, but not STAT5, in the esophageal epithelium, with highest levels in steroid-refractory EoE patients. PMID:21819482

  11. 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

  12. Nuclear EGFRvIII resists hypoxic microenvironment induced apoptosis via recruiting ERK1/2 nuclear translocation.

    PubMed

    Xie, Hui; Yang, Jinfeng; Xing, Wenjing; Dong, Yucui; Ren, Huan

    2016-02-01

    Glioblastoma (GBM) is the most aggressive type of primary brain tumor. Its interaction with the tumor microenvironment promotes tumor progression. Furthermore, GBM bearing expression of EGFRvIII displays more adaptation to tumor microenvironment related stress. But the mechanisms were poorly understood. Here, we presented evidence that in the human U87MG glioblastoma tumor model, EGFRvIII overexpression led aberrant kinase activation and nuclear translocation of EGFRvIII/ERK1/2 under hypoxia, which induced growth advantage by resisting apoptosis. Additionally, EGFRvIII defective in nuclear entry impaired this capacity in hypoxia adaptation, and partially interrupted ERK1/2 nuclear translocation. Pharmacology or genetic interference ERK1/2 decreased hypoxia resistance triggered by EGFRvIII expression, but not EGFRvIII nuclear translocation. In summary, this study identified a novel role for EGFRvIII in hypoxia tolerance, supporting an important link between hypoxia and subcellular localization alterations of the receptor. PMID:26742423

  13. Methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve injury model.

    PubMed

    Okada, Kiyoshi; Tanaka, Hiroyuki; Temporin, Ko; Okamoto, Michio; Kuroda, Yusuke; Moritomo, Hisao; Murase, Tsuyoshi; Yoshikawa, Hideki

    2010-04-01

    Methylcobalamin is a vitamin B12 analog and is necessary for the maintenance of the nervous system. Although some previous studies have referred to the effects of methylcobalamin on neurons, the precise mechanism of this effect remains obscure. Here we show that methylcobalamin at concentrations above 100 nM promotes neurite outgrowth and neuronal survival and that these effects are mediated by the methylation cycle, a metabolic pathway involving methylation reactions. We also demonstrate that methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle. In a rat sciatic nerve injury model, continuous administration of high doses of methylcobalamin improves nerve regeneration and functional recovery. Therefore, methylcobalamin may provide the basis for better treatments of nervous disorders through effective systemic or local delivery of high doses of methylcobalamin to target organs. PMID:20045411

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

    PubMed

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

    2014-01-17

    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. PMID:24342621

  15. Phosphorylation/dephosphorylation of human SULT4A1: role of Erk1 and PP2A.

    PubMed

    Mitchell, Deanne J; Butcher, Neville J; Minchin, Rodney F

    2011-01-01

    SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr(11) residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr(11) by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bβ was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase. PMID:20920535

  16. 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

  17. 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

  18. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors.

    PubMed

    Daulhac, L; Kowalski-Chauvel, A; Pradayrol, L; Vaysse, N; Seva, C

    1999-07-16

    We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. PMID:10400698

  19. Neuroprotective effects of 3,6'-disinapoyl sucrose through increased BDNF levels and CREB phosphorylation via the CaMKII and ERK1/2 pathway.

    PubMed

    Hu, Yuan; Liu, Ming-Yue; Liu, Ping; Dong, XianZhe; Boran, Aislyn D W

    2014-08-01

    3,6'-Disinapoyl sucrose (DISS) is an oligosaccharide ester natural product originating from the root of wild Polygala tenuifolia. Our previous reports suggested that DISS can have neuroprotective effects and antidepressive activity in rats, at least in part, by increased expression of cyclic AMP response element (CRE)-binding protein (CREB) and its downstream target protein, brain-derived neurotrophic factor (BDNF). The aim of the present study was to explore the mechanism of DISS-modulated BDNF and CREB expression. In this study, we confirmed its neuroprotective effect by showing that DISS, at concentrations above 30 μM, could promote the neuron cell viability and protected the glutamate and H2O2-induced toxicity in the human neuroblastoma (SH-SY5Y) cell line. DISS treatment also increased acute (from 15 to 30 min) BDNF expression and CREB phosphorylation in a dose-dependent manner. Pharmacological inhibition of mitogen-activated protein kinase 1 (ERK1/2), CaMKII, and Trk (with U0126, KN93, or K252a, respectively) partially attenuated the stimulatory effect of DISS on phospho-CREB and BDNF expression; however, it was not inhibited by pharmacological inhibition of PKA or PI3K (with H89 and LY294002, respectively). The results are consistent with the effects of DISS on CRE-directed gene transcription, as U0126 and KN-93 treatment also blocked the DISS-induced expression of the CRE-luciferase reporter gene. The results from the present study suggest that DISS-mediated regulation of BDNF gene expression is associated with CREB-mediated transcription of BDNF and upstream activation of ERK1/2 and CaMKII. Finally, DISS may exert neuroprotective and antidepressant effects through these signaling pathways in neuronal cells. PMID:24488601

  20. PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling.

    PubMed

    Liu, Heng-Jia; Ooms, Lisa M; Srijakotre, Nuthasuda; Man, Joey; Vieusseux, Jessica; Waters, JoAnne E; Feng, Yue; Bailey, Charles G; Rasko, John E J; Price, John T; Mitchell, Christina A

    2016-08-12

    PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21(WAF1/CIP1) PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stable PREX1 knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression. PMID:27358402

  1. The positive allosteric modulator of α7 nicotinic acetylcholine receptors, 3-furan-2-yl-N-p-tolyl-acrylamide, enhances memory processes and stimulates ERK1/2 phosphorylation in mice.

    PubMed

    Targowska-Duda, Katarzyna M; Wnorowski, Artur; Budzynska, Barbara; Jozwiak, Krzysztof; Biala, Grazyna; Arias, Hugo R

    2016-04-01

    To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs), improves memory processes, passive avoidance tests were conducted in male mice after acute and chronic treatments. To determine the neuronal mechanisms underlying the promnesic activity elicited by PAM-2, the effect of this ligand on α7 nAChR up-regulation and ERK1/2 phosphorylation was assessed in the hippocampus and prefrontal cortex. The results indicate that: (1) PAM-2 improves memory acquisition/consolidation after acute treatment (Day 2) and memory consolidation after chronic treatment (Day 22). Although no effect was observed on α7 nAChR up-regulation, the chronic, but not acute, PAM-2 treatment increases ERK1/2 kinase phosphorylation, (2) the promnesic activity of PAM-2 was inhibited by methyllycaconitine, a selective α7-antagonist, confirming the role of α7 nAChRs, (3) a synergistic (acute) effect was observed between inactive doses of PAM-2 (0.1mg/kg) and DMXBA (0.3mg/kg), a selective α7-agonist, and (4) PAM-2 reversed the memory impairment elicited by scopolamine, a muscarinic antagonist. The results demonstrate that PAM-2 presents promnesic activity mediated by α7 nAChRs, and is able to trigger ERK1/2 phosphorylation only after chronic treatment. PMID:26778787

  2. 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

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

    PubMed

    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

    2016-03-15

    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

  4. 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

  5. Saponins from the roots of Platycodon grandiflorum suppresses TGFβ1-induced epithelial-mesenchymal transition via repression of PI3K/Akt, ERK1/2 and Smad2/3 pathway in human lung carcinoma A549 cells.

    PubMed

    Choi, Jae Ho; Hwang, Yong Pil; Kim, Hyung Gyun; Khanal, Tilak; Do, Minh Truong; Jin, Sun Woo; Han, Hwa Jeong; Lee, Hyun Sun; Lee, Young Chun; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-01-01

    Transforming growth factor β (TGFβ) is a multifunctional cytokine that induces growth arrest, tissue fibrosis, and epithelial-mesenchymal transition (EMT) through activation of Smad and non-Smad signaling pathways. EMT is the differentiation switch by which polarized epithelial cells differentiate into contractile and motile mesenchymal cells. Our previous studies have shown that saponins from the roots of Platycodon grandiflorum (CKS) have antiinflammatory, antioxidant, antimetastatic, and hepatoprotective effects. In this study, we investigated the inhibitory effect of CKS on TGFβ1-induced alterations characteristic of EMT in human lung carcinoma A549 cells. We found that CKS-treated cells displayed inhibited TGFβ1-mediated E-cadherin downregulation and Vimentin upregulation and also retained epithelial morphology. Furthermore, TGFβ1-increased Snail expression, a repressor of E-cadherin and an inducer of the EMT, was reduced by CKS. CKS inhibited TGFβ1-induced phosphorylation of Akt, ERK1/2, and glycogen synthase kinase-3β (GSK-3β). Inhibition of PI3K/Akt and ERK1/2 also blocked TGFβ1-induced GSK-3β phosphorylation and Snail activation. Furthermore, TGFβ1-increased Snail expression was reduced by selective inhibitors of Akt and ERK1/2. Moreover, CKS treatment attenuated TGFβ1-induced Smad2/3 phosphorylation and upregulated Smad7 expression. These results indicate that pretreatment with the CKS inhibits the TGFβ1-induced EMT through PI3K/Akt, ERK1/2, GSK-3β and Smad2/3 in human lung carcinoma cells. PMID:24341702

  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. 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

  8. A New Paradigm for MAPK: Structural Interactions of hERK1 with Mitochondria in HeLa Cells

    PubMed Central

    Galli, Soledad; Jahn, Olaf; Hitt, Reiner; Hesse, Doerte; Opitz, Lennart; Plessmann, Uwe; Urlaub, Henning; Poderoso, Juan Jose; Jares-Erijman, Elizabeth A.; Jovin, Thomas M.

    2009-01-01

    Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells. PMID:19847302

  9. 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…

  10. 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...

  11. 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

  12. 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

  13. TPL-2-ERK1/2 signaling promotes host resistance against intracellular bacterial infection by negative regulation of type I IFN production.

    PubMed

    McNab, Finlay W; Ewbank, John; Rajsbaum, Ricardo; Stavropoulos, Evangelos; Martirosyan, Anna; Redford, Paul S; Wu, Xuemei; Graham, Christine M; Saraiva, Margarida; Tsichlis, Philip; Chaussabel, Damien; Ley, Steven C; O'Garra, Anne

    2013-08-15

    Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading cause of mortality and morbidity worldwide, causing ≈ 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL-12, IL-1, and TNF-α, as well as IFN-γ and CD4(+) Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I IFN have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL-10 is known to inhibit the immune response to M. tuberculosis in murine models through the negative regulation of key proinflammatory cytokines and the subsequent Th1 response. We show in this study, using a combination of transcriptomic analysis, genetics, and pharmacological inhibitors, that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I IFN production. The TPL-2-ERK1/2 signaling pathway regulated production by macrophages of several cytokines important in the immune response to M. tuberculosis as well as regulating induction of a large number of additional genes, many in a type I IFN-dependent manner. In the absence of TPL-2 in vivo, excess type I IFN promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I IFN may promote susceptibility to this important disease. PMID:23842752

  14. fundTPL-2 – ERK1/2 Signaling Promotes Host Resistance against Intracellular Bacterial Infection by Negative Regulation of Type I Interferon Production3

    PubMed Central

    McNab, Finlay W.; Ewbank, John; Rajsbaum, Ricardo; Stavropoulos, Evangelos; Martirosyan, Anna; Redford, Paul S.; Wu, Xuemei; Graham, Christine M.; Saraiva, Margarida; Tsichlis, Philip; Chaussabel, Damien; Ley, Steven C.; O’Garra, Anne

    2013-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality and morbidity worldwide, causing approximately 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL-12, IL-1 and TNF-α, as well as IFN-γ and CD4+ Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I interferon have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL-10 is known to inhibit the immune response to Mtb in murine models through the negative regulation of key pro-inflammatory cytokines and the subsequent Th1 response. We show here, using a combination of transcriptomic analysis, genetics and pharmacological inhibitors that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I interferon production. The TPL-2-ERK1/2 signalling pathway regulated production by macrophages of several cytokines important in the immune response to Mtb as well as regulating induction of a large number of additional genes, many in a type I IFN dependent manner. In the absence of TPL-2 in vivo, excess type I interferon promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I interferon may promote susceptibility to this important disease. PMID:23842752

  15. 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

  16. 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. PMID:25785932

  17. 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

  18. 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. PMID:26991852

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

    PubMed Central

    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 Ca2+ 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 Ca2+ 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 Ca2+ 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

  20. A Pilot Study of Quantitative Loop-mediated Isothermal Amplification-guided Target Therapies for Hospital-acquired Pneumonia

    PubMed Central

    Wang, Fang; Li, Ran; Shang, Ying; Wang, Can; Wang, Guo-Qing; Zhou, De-Xun; Yang, Dong-Hong; Xi, Wen; Wang, Ke-Qiang; Bao, Jing; Kang, Yu; Gao, Zhan-Cheng

    2016-01-01

    Background: It is important to achieve the definitive pathogen identification in hospital-acquired pneumonia (HAP), but the traditional culture results always delay the target antibiotic therapy. We assessed the method called quantitative loop-mediated isothermal amplification (qLAMP) as a new implement for steering of the antibiotic decision-making in HAP. Methods: Totally, 76 respiratory tract aspiration samples were prospectively collected from 60 HAP patients. DNA was isolated from these samples. Specific DNA fragments for identifying 11 pneumonia-related bacteria were amplified by qLAMP assay. Culture results of these patients were compared with the qLAMP results. Clinical data and treatment strategies were analyzed to evaluate the effects of qLAMP results on clinical data. McNemar test and Fisher's exact test were used for statistical analysis. Results: The detection of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Stenotrophomonas maltophilia, Streptococcus pneumonia, and Acinetobacter baumannii by qLAMP was consistent with sputum culture (P > 0.05). The qLAMP results of 4 samples for Haemophilus influenzae, Legionella pneumophila, or Mycoplasma pneumonia (MP) were inconsistent with culture results; however, clinical data revealed that the qLAMP results were all reliable except 1 MP positive sample due to the lack of specific species identified in the final diagnosis. The improvement of clinical condition was more significant (P < 0.001) in patients with pathogen target-driven therapy based on qLAMP results than those with empirical therapy. Conclusion: qLAMP is a more promising method for detection of pathogens in an early, rapid, sensitive, and specific manner than culture. PMID:26830989

  1. Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia.

    PubMed

    Gotoh, Kensei; Nishimura, Naoko; Ohshima, Yasunori; Arakawa, Yasuko; Hosono, Haruki; Yamamoto, Yasuto; Iwata, Yasushi; Nakane, Kazumasa; Funahashi, Keiji; Ozaki, Takao

    2012-10-01

    Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1-14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia. PMID:22370920

  2. Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

    SciTech Connect

    Holmstroem, Therese E.; Mattsson, Charlotte L.; Faelting, Johanna M.; Nedergaard, Jan

    2008-11-15

    Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical {zeta} isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Goe6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation ({sup 3}H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.

  3. Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    PubMed Central

    Yoon, Yae Jin; Kim, Dae-Kyum; Yoon, Chang Min; Park, Jaesung; Kim, Yoon-Keun; Roh, Tae-Young; Gho, Yong Song

    2014-01-01

    Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference–mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases. PMID:25502753

  4. 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.

  5. Adiponectin Upregulates MiR-133a in Cardiac Hypertrophy through AMPK Activation and Reduced ERK1/2 Phosphorylation

    PubMed Central

    Guan, Yuqing; Wang, Lei; Wang, Shuya; Li, Yueyan; Fu, Ying; Gao, Xiaoyuan; Su, Guohai

    2016-01-01

    Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II) induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF) and brain natriuretic peptide (BNP), also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd). Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs) with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA) cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1) mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF), a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in cardiomyocytes

  6. Adiponectin Upregulates MiR-133a in Cardiac Hypertrophy through AMPK Activation and Reduced ERK1/2 Phosphorylation.

    PubMed

    Li, Ying; Cai, Xiaojun; Guan, Yuqing; Wang, Lei; Wang, Shuya; Li, Yueyan; Fu, Ying; Gao, Xiaoyuan; Su, Guohai

    2016-01-01

    Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II) induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF) and brain natriuretic peptide (BNP), also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd). Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs) with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA) cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1) mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF), a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in cardiomyocytes

  7. Differential Targeting of SLC30A10/ZnT10 Heterodimers to Endolysosomal Compartments Modulates EGF-Induced MEK/ERK1/2 Activity.

    PubMed

    Zhao, Yitong; Feresin, Rafaela G; Falcon-Perez, Juan M; Salazar, Gloria

    2016-03-01

    The solute carrier 30A (SLC30A) family of zinc exporters transports zinc into the lumen of intracellular organelles in order to prevent zinc toxicity. We reported that formation of tyrosine dimers is required for ZnT3 (zinc transporter 3) zinc transport activity and targeting to synaptic-like microvesicles (SLMVs) in PC12 cells and the formation of ZnT3/ZnT10 heterodimers. Here, we focused on ZnT10 to determine the role of heterodimerization in the sorting of ZnTs in the endolysosomal pathway. Using cell fractionation, immunoprecipitation and immunofluorescence approaches, we found that ZnT10 resides in transferrin receptor and Rab5-positive endosomes and forms covalent heterodimers and oligomers with ZnT2, ZnT3 and ZnT4. The interaction of ZnT10 with ZnT3, mediated by dityrosine bonds, was unable to target ZnT10 into SLMVs in vitro or into synaptic vesicles isolated from mouse brain in vivo. However, ZnT3/ZnT10 heterodimers regulate epidermal growth factor receptor (EGF-R) signaling by increasing the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK1/2), but not EGF-R, C-Raf or Akt phosphorylation in response to EGF. Further, mutation of tyrosine 4 in ZnT10 reduced ZnT3/ZnT10 dityrosine-mediated heterodimerization and zinc transport, as well as MEK and ERK1/2 phosphorylation, which were also reduced by the zinc chelator TPEN. Phosphorylation of these kinases is likely to occur in the cytosol as no differences in phosphorylation were observed in membrane fractions of control and ZnT3/ZnT10-expressing cells. We propose that ZnT10 plays a role in signal transduction, which is mediated by homo and heterodimerization with other ZnTs. PMID:26728129

  8. Fibroblast Growth Factor–2 Promotes Catabolism Via FGFR1–Ras–Raf–MEK1/2–ERK1/2 Axis That Coordinates With the PKCδ Pathway in Human Articular Chondrocytes

    PubMed Central

    Yan, Dongyao; Chen, Di; Im, Hee-Jeong

    2013-01-01

    Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes. PMID:22488450

  9. Hesperidin Induces Paraptosis Like Cell Death in Hepatoblatoma, HepG2 Cells: Involvement of ERK1/2 MAPK

    PubMed Central

    Yumnam, Silvia; Park, Hyeon Soo; Kim, Mun Ki; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Lee, Won Sup; Kim, Eun Hee; Cho, Jae Hyeon; Shin, Sung Chul; Kim, Gon Sup

    2014-01-01

    Hesperidin, a natural flavonoid abundantly present in Citrus is known for its anti-cancer, anti-oxidant and anti-inflammatory properties. In this study we examined the effect of hesperidin on HepG2 cells. HepG2 cells treated with various concentration of hesperidin undergo a distinct type of programed cell death. Cytoplasmic vacuolization, mitochondria and endoplasmic reticulum swelling and uncondensed chromatin were observed in hesperidin treated cells. DNA electrophoresis show lack of DNA fragmentation and western blot analysis demonstrates lack of caspase activation and PARP cleavage. It was observed that hesperidin induced cell death is nonautophagic and also activate mitogen activated protein kinase ERK1/2. Taken together, the data indicate that hesperidin induces paraptosis like cell death in HepG2 cells with the activation of ERK1/2. Thus our finding suggests that hesperidin inducing paraptosis may offer an alternative tool in human liver carcinoma therapy. PMID:24977707

  10. A novel pro-inflammatory protein of Streptococcus suis 2 induces the Toll-like receptor 2-dependent expression of pro-inflammatory cytokines in RAW 264.7 macrophages via activation of ERK1/2 pathway

    PubMed Central

    Zhang, Qiang; Yang, Yujie; Yan, Shuxian; Liu, Jiantao; Xu, Zhongmin; Yu, Junping; Song, Yajing; Zhang, Anding; Jin, Meilin

    2015-01-01

    Streptococcus suis 2 is an important swine pathogen and an emergent zoonotic pathogen. Excessive inflammation caused by S. suis is responsible for the high levels of early mortality observed in septic shock-like syndrome cases. However, the mechanisms through which S. suis 2 (SS2) causes excessive inflammation remain unclear. Thus, this study aimed to identify novel pro-inflammatory mediators that play important roles in the development of therapies against SS2 infection. In this study, the novel pro-inflammatory protein HP0459, which was encoded by the SSUSC84_0459 gene, was discovered. The stimulation of RAW 264.7 macrophages with recombinant HP0459 protein induced the expression of pro-inflammatory cytokines (IL-1β, MCP-1 and TNF-α). Compared with the wild-type (WT) strain, the isogenic knockout of HP0459 in SS2 led to reduced production of pro-inflammatory cytokines in RAW264.7 macrophages and in vivo. The pro-inflammatory activity of HP0459 was significantly reduced by an antibody against Toll-like receptor 2 (TLR2) in RAW264.7 macrophages and was lower in TLR2-deficient (TLR2-/-) macrophages than in WT macrophages. Furthermore, specific inhibitors of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathways significantly decreased the HP0459-induced pro-inflammatory cytokine production, and a western blot assay showed that HP0459 stimulation induced the activation of the ERK1/2 pathway. Taken together, our data indicate that HP0459 is a novel pro-inflammatory mediator of SS2 and induces TLR2-dependent pro-inflammatory activity in RAW264.7 macrophages through the ERK1/2 pathway. PMID:25806027

  11. 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

  12. Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells.

    PubMed

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

    2011-11-01

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

  13. Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2

    PubMed Central

    Parkinson, Eric Kenneth

    2013-01-01

    The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)—eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)—inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo. PMID:23892603

  14. Radiation-Induced c-Jun Activation Depends on MEK1-ERK1/2 Signaling Pathway in Microglial Cells

    PubMed Central

    Deng, Zhiyong; Sui, Guangchao; Rosa, Paulo Mottin; Zhao, Weiling

    2012-01-01

    Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation. PMID:22606284

  15. 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. PMID:26565022

  16. 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

  17. 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

  18. Bisphenol A, an environmental estrogen-like toxic chemical, induces cardiac fibrosis by activating the ERK1/2 pathway.

    PubMed

    Hu, Yingying; Zhang, Li; Wu, Xianxian; Hou, Liangyu; Li, Zhange; Ju, Jin; Li, Qian; Qin, Wei; Li, Jiamin; Zhang, Qingwei; Zhou, Tong; Zhang, Longyin; Xu, Chaoqian; Fang, Zhiwei; Zhang, Yong

    2016-05-27

    Bisphenol A (BPA) is a widely studied typical endocrine-disrupting chemical. The present study aimed to verify whether BPA could induce proliferation of cardiac fibroblasts and collagen production leading to cardiac interstitial fibrosis. After exposure to BPA for 30 consecutive days, decreased cardiac function was observed in rats using echocardiography, and the deposition of collagen was detected by Masson's trichrome staining and electron microscope. BPA remarkably stimulated proliferation and migration of cultured cardiac fibroblasts and collagen production in a concentration-dependent manner, as revealed by MTT, wound healing assay and collagen assay. Meanwhile, BPA treatment also enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In contrast, pretreatment with estrogen receptor inhibitor ICI182780 or ERK inhibitor PD98059 prevented the enhanced phosphorylation of ERK1/2, and subsequently inhibited the up-regulation of transforming growth factor-β1 (TGF-β1) expression induced by BPA. As a consequence, these inhibitors also decreased proliferation and collagen production, as well as the fibrosis-related genes expression. Taken together, our results indicated that BPA may act as a promoting factor in proliferative process and collagen production of cardiac fibroblasts via activating ERK1/2. PMID:27025157

  19. Radial Spoke Protein 3 Is a Mammalian Protein Kinase A-anchoring Protein That Binds ERK1/2*

    PubMed Central

    Jivan, Arif; Earnest, Svetlana; Juang, Yu-Chi; Cobb, Melanie H.

    2009-01-01

    Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIα and RIIβ, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes. PMID:19684019

  20. Interleukin-1β promotes the neurogenesis of carotid bodies by stimulating the activation of ERK1/2.

    PubMed

    Xue, Fen; Liu, Ling; Fan, Juan; He, ShanShan; Li, Rui; Peng, Zheng-Wu; Wang, Bai-Ren

    2015-12-01

    The carotid body (CB) is a complex sensory organ that functions to sense homeostatic O2 in the blood. Previous studies have shown that CBs express interleukin (IL)-1 receptor type I and that the chemosensitivity of CBs is increased following stimulation with pro-inflammatory cytokines. However, the effects of pro-inflammatory cytokines, such as IL-1β, on the neurogenesis of CB are unclear. Thus, in this study, we aimed to assess the effects of IL-1β and intermittent hypobaric hypoxia (IHH) plus IL-1β on the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, tyrosine hydroxylase (TH) and the expression of nestin, a well-established stem cell marker in the nervous system. The results showed that TH, nestin expression and ERK1/2 phosphorylation were increased in the rat CB following intraperitoneal injection of IL-1β. Moreover, IL-1β had additive effects on IHH. These results suggested that the plasticity of CB was increased following treatment with IL-1β and that ERK1/2 may be involved in neurogenic signaling in CBs. PMID:26327233

  1. 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

  2. Regulator of G protein signalling 14 attenuates cardiac remodelling through the MEK-ERK1/2 signalling pathway.

    PubMed

    Li, Ying; Tang, Xiao-Hong; Li, Xiao-Hui; Dai, Hai-Jiang; Miao, Ru-Jia; Cai, Jing-Jing; Huang, Zhi-Jun; Chen, Alex F; Xing, Xiao-Wei; Lu, Yao; Yuan, Hong

    2016-07-01

    In the past 10 years, several publications have highlighted the role of the regulator of G protein signalling (RGS) family in multiple diseases, including cardiovascular diseases. As one of the multifunctional family members, RGS14 is involved in various biological processes, such as synaptic plasticity, cell division, and phagocytosis. However, the role of RGS14 in cardiovascular diseases remains unclear. In the present study, we used a genetic approach to examine the role of RGS14 in pathological cardiac remodelling in vivo and in vitro. We observed that RGS14 was down-regulated in human failing hearts, murine hypertrophic hearts, and isolated hypertrophic cardiomyocytes. Moreover, the extent of aortic banding-induced cardiac hypertrophy and fibrosis was exacerbated in RGS14 knockout mice, whereas RGS14 transgenic mice exhibited a significantly alleviated response to pressure overload. Furthermore, research of the underlying mechanism revealed that the RGS14-dependent rescue of cardiac remodelling was attributed to the abrogation of mitogen-activated protein kinase (MEK)-extracellular signal-regulated protein kinase (ERK) 1/2 signalling. The results showed that constitutive activation of MEK1 nullified the cardiac protection in RGS14 transgenic mice, and inhibition of MEK-ERK1/2 by U0126 reversed RGS14 deletion-related hypertrophic aggravation. These results demonstrated that RGS14 attenuated the development of cardiac remodelling through MEK-ERK1/2 signalling. RGS14 exhibited great potential as a target for the treatment of pathological cardiac remodelling. PMID:27298141

  3. 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.

  4. Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

    PubMed Central

    Murray, Thomas V.A.; Smyrnias, Ioannis; Schnelle, Moritz; Mistry, Rajesh K.; Zhang, Min; Beretta, Matteo; Martin, Daniel; Anilkumar, Narayana; de Silva, Shana M.; Shah, Ajay M.; Brewer, Alison C.

    2015-01-01

    Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell

  5. 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

  6. Attenuation of Magnesium Sulfate on CoCl₂-Induced Cell Death by Activating ERK1/2/MAPK and Inhibiting HIF-1α via Mitochondrial Apoptotic Signaling Suppression in a Neuronal Cell Line.

    PubMed

    Huang, Chih-Yang; Hsieh, You-Liang; Ju, Da-Tong; Lin, Chien-Chung; Kuo, Chia-Hua; Liou, Yi-Fan; Ho, Tsung-Jung; Tsai, Chang-Hai; Tsai, Fuu-Jen; Lin, Jing-Ying

    2015-08-31

    Magnesium sulfate (MgSO₄) ameliorates hypoxia/ischemia-induced neuronal apoptosis in a rat model. This study aimed to investigate the mechanisms governing the anti-apoptotic effect of MgSO₄ on cobalt chloride (CoCl₂)-exposed NB41A3 mouse neuroblastoma cells. MgSO₄ increased the viability of NB41A3 cells treated with CoCl₂ in a dose-dependent manner. MgSO₄ treatment was shown to lead to an increase in the anti-apoptotic Bcl-2 family proteins, with a concomitant decrease in the pro-apoptotic proteins. MgSO₄ also attenuated the CoCl₂-induced disruption of mitochondrial membrane potential (ΔΨ(m)) and reduced the release of cytochrome c form the mitochondria to the cytosol. Furthermore, exposure to CoCl₂ caused activation of the hypoxia-inducible factor 1α (HIF-1α). On the other hand, MgSO₄ markedly reduced CoCl₂-induced HIF-1α activation and suppressed HIF-1α downstream protein BNIP3. MgSO₄ treatment induced ERK1/2 activation and attenuated CoCl₂-induced activation of p38 and JNK. Addition of the ERK1/2 inhibitor U0126 significantly reduced the ability of MgSO₄ to protect neurons from CoCl₂-induced mitochondrial apoptotic events. However, incubation of cultures with the p38 and JNK inhibitors did not significantly affect MgSO₄-mediated neuroprotection. MgSO₄ appears to suppress CoCl₂-induced NB41A3 cell death by activating ERK1/2/ MAPK pathways, which further modulates the role of Bcl-2 family proteins and mitochondria in NB41A3 cells. Our data suggest that MgSO₄ may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways. PMID:26211648

  7. 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.

  8. 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. PMID:25772140

  9. 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.

  10. Distinct effect of CacyBP/SIP on the ERK1/2-CREB-BDNF pathway in undifferentiated and differentiated neuroblastoma NB2a cells.

    PubMed

    Rosińska, Sara; Leśniak, Wiesława; Filipek, Anna

    2016-07-01

    CacyBP/SIP, a protein expressed to high extent in the brain, has been shown to act as ERK1/2 phosphatase in vitro and in cultured cells. It has been demonstrated recently that CacyBP/SIP can modulate the activity of some transcription factors in neurons and glioma cells. In the present work we have examined the effect of CacyBP/SIP overexpression and silencing on the phosphorylation/activity of ERK1/2 (pERK1/2) and CREB (pCREB) and on the level of BDNF mRNA in differentiated and undifferentiated neuroblastoma NB2a cells. We have shown that in undifferentiated cells the amount of pERK1/2 decreased upon CacyBP/SIP overexpression. Further studies have shown that the activity of CREB and the level of BDNF mRNA, downstream effectors of the ERK1/2 signaling pathway, also depended on the CacyBP/SIP level and strictly matched the level of pERK1/2. Interestingly, in differentiated NB2a cells, overexpression of CacyBP/SIP appeared to have a distinct effect on the pERK1/2 level from that observed in undifferentiated cells. Subsequent studies have revealed that distinct function of CacyBP/SIP in undifferentiated and differentiated NB2a cells might be due to changes in its posttranslational modifications and protein ligands. Altogether, our studies suggest that CacyBP/SIP is involved in the ERK1/2-CREB-BDNF pathway and that it might regulate this pathway depending on the stage of NB2a cell differentiation. PMID:27180052

  11. PI3K and ERK1/2 kinase inhibition potentiate protease inhibitor to attenuate allergen induced Th2 immune response in mouse.

    PubMed

    Saw, Sanjay; Arora, Naveen

    2016-04-01

    Proteases affect immune response by activating PI3K, ERK1/2 and p38 kinase. In present study, therapeutic effect of PI3K, ERK1/2 and p38 kinase inhibitor in combination with serine protease inhibitor was evaluated in cockroach extract (CE) induced airway inflammatory disease. Mice were sensitized on day 0, 7 and 14 and challenged on day 27, 28 and 29 with CE. Mice were given PI3K, ERK1/2 and the p38 kinase inhibitor (iPI3K, iERK1/2 and the ip38) alone or with serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1h before challenge. On day 30 airway resistance of mice were determined and euthanized to collect blood, BAL fluid and lung for analysis. CE immunized mice showed PI3K, ERK1/2 and p38 kinase activation, increased airway resistance, cellular infiltration, Th2 cytokines IgE and IgG1. AEBSF given to mice reduced the CE induced allergic response. AEBSF given in combination of iPI3K/iERK1/2 reduced cellular infiltration in lungs. Furthermore, iPI3K/iERK1/2 with AEBSF significantly reduced the CE induced Th2 cytokines in comparison to monotherapy of kinase inhibitor and AEBSF (P<0.05). The combination of iPI3K/iERK1/2 with AEBSF enhanced IL-12 level that could further provide a mean of Th2 reduction. Best effect in reduction of allergic response in mice was observed on administration of AEBSF with iPI3K. Conclusively, the combination of PI3K kinase inhibitor with AEBSF reduced allergen induced airway response and has therapeutic potential for add-on therapy in allergic airway disease. PMID:26905476

  12. 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. PMID:25939061

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

    PubMed Central

    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; Cantarini, Mireille; Barrett, J. Carl; Jänne, Pasi A.; Oxnard, Geoffrey R.

    2015-01-01

    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 T790M prior to treatment, but at resistance three molecular subtypes emerged: 6 cases acquired the C797S mutation, 5 cases maintained the T790M mutation but did not acquire the C797S mutation, and 4 cases lost the T790M mutation despite detecting 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 able to overcome resistance mediated by EGFR C797S. PMID:25939061

  14. Bacillus anthracis endospores regulate ornithine decarboxylase and inducible nitric oxide synthase through ERK1/2 and p38 mitogen-activated protein kinases.

    PubMed

    Porasuphatana, Supatra; Cao, Guan-Liang; Tsai, Pei; Tavakkoli, Fatemeh; Huwar, Theresa; Baillie, Les; Cross, Alan S; Shapiro, Paul; Rosen, Gerald M

    2010-12-01

    Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide ((•)NO) from inducible nitric oxide synthase (NOS2) by competing for L-arginine, producing L-ornithine at the expense of (•)NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of L-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores' germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms. PMID:20440620

  15. 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. PMID:26189563

  16. 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. PMID:21115804

  17. Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells.

    PubMed

    Joo, Donghyun; Woo, Jong Soo; Cho, Kwang-Hyun; Han, Seung Hyun; Min, Tae Sun; Yang, Deok-Chun; Yun, Cheol-Heui

    2016-04-01

    Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225]. PMID:26879318

  18. β-Arrestin 2-dependent activation of ERK1/2 is required for ADP-induced paxillin phosphorylation at Ser83 and microglia chemotaxis

    PubMed Central

    Lee, Sang-Hyun; Hollingsworth, Ryan; Kwon, Hyeok-Yil; Lee, Narae; Chung, Chang Y.

    2014-01-01

    Microglia play crucial roles in increased inflammation in the CNS upon brain injuries and diseases. Extracellular ADP has been reported to induce microglia chemotaxis and membrane ruffle formation through P2Y12 receptor. In this study, we examined the role of ERK1/2 activation in ADP-induced microglia chemotaxis. ADP stimulation increases the phosphorylation of ERK1/2 and paxillin phosphorylation at Tyr31 and Ser83. Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser83 and the retraction of membrane ruffles, causing inefficient chemotaxis. Close examination of dynamics of focal adhesion formation with GFP-paxillin revealed that the disassembly of focal adhesions in U0126-treated cells was significantly impaired. Depletion of β-Arr2 with shRNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser83, indicating that β-Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and β-Arr2 were translocated and co-localized at focal contacts in the newly forming lamellipodia. Examination of kinetics and rate constant of paxillin formation and disassembly revealed that the phosphorylation of paxillin at Tyr31 by c-Src appears to be involved in adhesion formation upon ADP stimulation while Ser83 required for adhesion disassembly. PMID:22638989

  19. Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

    PubMed Central

    Joo, Donghyun; Woo, Jong Soo; Cho, Kwang-Hyun; Han, Seung Hyun; Min, Tae Sun; Yang, Deok-Chun; Yun, Cheol-Heui

    2016-01-01

    Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225] PMID:26879318

  20. Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing

    PubMed Central

    Warren, D T; Tajsic, T; Porter, L J; Minaisah, R M; Cobb, A; Jacob, A; Rajgor, D; Zhang, Q P; Shanahan, C M

    2015-01-01

    Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs. PMID:25744025

  1. 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. PMID:27063575

  2. 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

  3. 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

  4. Angiopoietin-1 attenuates angiotensin II-induced ER stress in glomerular endothelial cells via a Tie2 receptor/ERK1/2-p38 MAPK-dependent mechanism.

    PubMed

    Bi, Xiao; Niu, Jianying; Ding, Wei; Zhang, Minmin; Yang, Min; Gu, Yong

    2016-06-15

    Research has indicated that endoplasmic reticulum (ER) stress in endothelial cells affects vascular pathologies and induces cellular dysfunction and apoptosis. Angiopoietin1 (Angpt1) has been shown to have therapeutic potential in some vascular diseases, including chronic kidney disease. This study showed that Angpt1 is a powerful factor that attenuated ER stress-induced cellular dysfunction and apoptosis in glomerular endothelial cells (GEnCs). Furthermore, Angpt1 significantly decreased the angiotensin II (Ang II)-induced expression of the ER stress response proteins GRP78, GRP94, p-PERK and CHOP. These results suggest that the Angpt1-mediated cellular protection may occur downstream of the ER stress response. In addition, both specific inhibitors and siRNAs for Tie2 reversed these changes, implying the importance of Tie2 receptor activation in the signalling pathways that prevent ER stress. The protective effects of Angpt1 are related to the activation of two downstream signalling pathways, ERK1/2 and p38 MAPK. The inhibition of these pathways with specific inhibitors, PD98059 and SB203580, respectively, partially increased the expression of chaperones that assist in folding proteins in the ER and reduce the protective effects of Angpt1. In conclusion, Angpt1 attenuated ER stress-induced cellular dysfunction and apoptosis via the Tie2 receptor/ERK1/2-p38 MAPK pathways in GEnCs. This study may provide insights into a novel underlying mechanism and a strategy for alleviating ER stress-induced injury. PMID:27033326

  5. Eicosapentaenoic acid induced SKOV-3 cell apoptosis through ERK1/2-mTOR-NF-κB pathways.

    PubMed

    Han, Lirong; Zhang, Yuanyuan; Meng, Meng; Cheng, Dai; Wang, Chunling

    2016-08-01

    Eicosapentaenoic acid (EPA), a typical kind of n-3 polyunsaturated fatty acids, has been considered to be a potent antitumor adjuvant. However, the mechanism related to EPA-induced SKOV-3 cell apoptosis has not been investigated. In this study, we elucidated the anticancer effect of EPA on SKOV-3 cells and its molecular mechanisms. The results of fluorescence microscopy showed that EPA induced typical apoptotic morphological features in SKOV-3 cells. Flow cytometric analysis indicated that EPA induced apoptosis of SKOV-3 cells through cells arrested at the S phase. Western blotting results showed that EPA could inhibit the phosphorylation of ERK1/2 and Akt, which restrained mammalian target of rapamycin (mTOR) phosphorylated. Simultaneously, EPA downregulated the phosphorylation status of mTOR, which may act as an upstream regulator of EPA-blocked nuclear factor κB (NF-κB) p65 translocation from the cytoplasm into the nucleus; the apoptotic mechanism of SKOV-3 cells induced by EPA was associated with the release of cytochrome c, Bax-to-Bcl-2 expression ratio, and activation of caspase-3 and caspase-9. The results suggested that EPA induced SKOV-3 cell apoptosis through ERK1/2, Akt-mTOR-NF-κB pathways. PMID:27176035

  6. Surface Chemical Gradient Affects the Differentiation of Human Adipose-Derived Stem Cells via ERK1/2 Signaling Pathway.

    PubMed

    Liu, Xujie; Shi, Shengjun; Feng, Qingling; Bachhuka, Akash; He, Wei; Huang, Qianli; Zhang, Ranran; Yang, Xing; Vasilev, Krasimir

    2015-08-26

    To understand the role of surface chemistry on cell behavior and the associated molecular mechanisms, we developed and utilized a surface chemical gradient of amine functional groups by carefully adjusting the gas composition of 1,7-octadiene (OD) and allylamine (AA) of the plasma phase above a moving substrate. The chemical gradient surface used in the present work shows an increasing N/C ratio and wettability from the OD side toward the AA side with no change in surface topography. Under standard culture conditions (with serum), human adipose-derived stem cells (hASCs) adhesion and spreading area increased toward the AA side of the gradient. However, there were no differences in cell behavior in the absence of serum. These results, supported by the trends in proteins adsorption on the gradient surface, demonstrated that surface chemistry affects the response of hASCs through cell-adhesive serum proteins, rather than interacting directly with the cells. The expression of p-ERK and the osteogenic differentiation increased toward the AA side of the gradient, while adipogenic differentiation decreased in the same direction; however, when the activation of ERK1/2 was blocked by PD98059, the levels of osteogenic or adipogenic differentiation on different regions of the chemical gradient were the same. This indicates that ERK1/2 may be an important downstream signaling pathway of surface chemistry directed stem cell fate. PMID:26237746

  7. 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. PMID:24259565

  8. Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism.

    PubMed

    Cubero, Francisco Javier; Nieto, Natalia

    2012-07-15

    Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease. PMID:22538404

  9. NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.

    PubMed

    Muñoz-Caro, Tamara; Lendner, Matthias; Daugschies, Arwid; Hermosilla, Carlos; Taubert, Anja

    2015-10-01

    Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum. PMID:26026247

  10. 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

  11. Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR

    PubMed Central

    Xu, Xiaohua; Balsiger, Robert; Tyrrell, Jean; Boyaka, Prosper N.; Tarran, Robert; Cormet-Boyaka, Estelle

    2015-01-01

    Background CFTR plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date. Methods The molecular mechanisms leading to down-regulation of CFTR by cigarette smoke were determined using pharmacologic inhibitors and silencing RNAs. Results Using human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by the lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke. Conversely, specific inhibitors of the JNK or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant NAC inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR. Conclusions These results show that the MEK/Erk1/2 MAPK pathway regulates plasma membrane CFTR in human airway cells. General Significance The MEK/Erk1/2 MAPK pathway should be considered as a target for strategies to maintain/restore CFTR expression in the lung of smokers. PMID:25697727

  12. 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

  13. 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. PMID:26906511

  14. DOPAMINE D2 RECEPTOR ACTIVATION LEADS TO AN UPREGULATION OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR VIA Gβγ-ERK1/2-DEPENDENT INDUCTION OF ZIF268

    PubMed Central

    Ahmadiantehrani, Somayeh; Ron, Dorit

    2013-01-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent growth factor essential to the development, survival, and function of dopaminergic neurons (Airaksinen and Saarma, 2002). The molecular mechanisms underlying GDNF expression remain elusive, thus, we set out to identify a signaling pathway that governs GDNF levels. We found that treatment of both differentiated dopaminergic-like SH-SY5Y cells and rat midbrain slices with the dopamine D2 receptor (D2R) agonist, quinpirole, triggered an increase in the expression of GDNF that was temporally preceded by an increase in the levels of Zif268, a DNA-binding transcription factor encoded by an immediate-early gene. Moreover, the D2R inhibitor raclopride blocked the increase of both GDNF and Zif268 expression following potassium-evoked dopamine release in SH-SY5Y cells. We used adenoviral delivery of small hairpin RNA (shRNA) targeting Zif268 to downregulate its expression and found that Zif268 is specifically required for the D2R-mediated upregulation of GDNF. Furthermore, the D2R-mediated induction of GDNF and Zif268 expression was dependent on Gβγ-mediated signaling and activation of extracellular signal-regulated kinase 1/2 (Erk1/2). Importantly, using chromatin immunoprecipitation (ChIP) assay, we identified a direct association of Zif268 with the GDNF promoter. These results suggest that D2R activation induces a Gβγ- and Erk1/2-dependent increase in the level of Zif268, which functions to directly upregulate the expression of GDNF. PMID:23373701

  15. 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

  16. Tribulosin suppresses apoptosis via PKC epsilon and ERK1/2 signaling pathway during hypoxia/reoxygenation in neonatal rat ventricular cardiac myocytes.

    PubMed

    Zhang, Shuang; Li, Hong; Yang, Shi-Jie

    2011-12-01

    Tribulosin (tigogenin 3-O-β-D-xylopyranosyl(1-2)-[β-D-xylopyranosyl (1-3)]-β-D-glucopyranosyl (1-4)-[a-L-rhamnopyranosyl(1-2)]-β-D-galactopyranoside), a component of gross saponins of Tribulus terrestris, has been shown to produce cytoprotective effects in heart. Yet, the precise mechanisms are not fully understood. We examined the mechanisms of tribulosin on myocardial protection. Ventricular myocytes were isolated from the heart of neonatal rats and were exposed to 3 h of hypoxia followed by 2 h reoxygenation. Apoptosis was induced by hypoxia/reoxygenation (H/R), and the expression of protein kinase C epsilon (PKCϵ) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) in cultured neonatal rat cardiac myocytes was detected. The results indicated that treatment with tribulosin in the culture medium protected cardiac myocytes against apoptosis induced by H/R. PKCϵ and ERK1/2 expression increased after pretreated with tribulosin. In the presence of PKCϵ inhibitor co-treated with tribulosin, the expression of ERK1/2 was decreased in H/R cardiac myocytes. While preconditioned with PD98059, ERK1/2 inhibitor, no effects on the expression of PKCϵ were detected. Tribulosin has protective effects on cardiac myocytes against apoptosis induced by H/R injury via PKCϵ and ERK1/2 signaling pathway. PMID:22115037

  17. Hydrogen sulfide promotes cell proliferation of oral cancer through activation of the COX2/AKT/ERK1/2 axis.

    PubMed

    Zhang, Shuai; Bian, Huan; Li, Xiaoxu; Wu, Huanhuan; Bi, Qingwei; Yan, Yingbin; Wang, Yixiang

    2016-05-01

    Hydrogen sulfide, the third gaseous transmitter, is one of the main causes of halitosis in the oral cavity. It is generally considered as playing a deleterious role in many oral diseases including oral cancer. However, the regulatory mechanisms involved in the effects of hydrogen sulfide on oral cancer growth remain largely unknown. In the present study, we investigated the underlying mechanisms through CCK-8 assay, EdU incorporation, real-time PCR, western blot and pathway blockade assays. Our results showed that hydrogen sulfide promoted oral cancer cell proliferation through activation of the COX2, AKT and ERK1/2 pathways in a dose-dependent manner. Blocking any of the three above pathways inhibited hydrogen sulfide-induced oral cancer cell proliferation. Meanwhile, blockade of COX2 by niflumic acid downregulated NaHS-induced p-ERK and p-AKT expression. Inactivation of the AKT pathway by GSK690693 significantly decreased NaHS‑induced p-ERK1/2 expression, and inhibition of the ERK1/2 pathway by U0126 markedly increased NaHS-induced p-AKT expression. Either the AKT or ERK1/2 inhibitor did not significantly alter the COX2 expression level. Our data revealed, for the first time, that hydrogen sulfide promotes oral cancer cell proliferation through activation of the COX2/AKT/ERK1/2 axis, suggesting new potential targets to eliminate the effect of hydrogen sulfide on the development of oral cancer. PMID:26987083

  18. 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

  19. Up-regulation of matrix metalloproteinases-2 and -9 via an Erk1/2/NF-κB pathway in murine mast cells infected with Toxoplasma gondii.

    PubMed

    Wang, M-F; Lu, C-Y; Lai, S-C

    2013-01-01

    Mast cells are key effectors in inflammation and contain proteinases that are released on activation. This study investigates associations between extracellular signal-regulated kinase (Erk)1/2, nuclear factor (NF)-κB, matrix metalloproteinase (MMP)-2 and MMP-9 in mast cells infected with Toxoplasma gondii tachyzoites. T. gondii infection led to increased mast cell degranulation. Phosphorylated (p)-Erk1/2 and p-NF-κB were increased significantly in mast cells infected with T. gondii. Pretreatment with the Erk kinase inhibitor PD98059 significantly decreased the expression of p-Erk1/2, p-NF-κB, MMP-2 and MMP-9. Treatment with MG132, an indirect NF-κB inhibitor, effectively reduced p-IκBα, p-NF-κB, MMP-2 and MMP-9 expression. Collectively, these data show that suppression of an Erk1/2/NF-κB signalling pathway caused a reduction in MMP-2 and -9 activities. Inhibiting this signalling pathway for MMP-2 and MMP-9 expression might offer a potential way to control early T. gondii infection. This pathway for the generation of MMP-2 and MMP-9 is important for mast cell secretion and the NF-κB/Erk1/2 signalling pathway may be key in MMP-2 and MMP-9 production in host defense against toxoplasmosis. PMID:23664424

  20. 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

  1. O-GlcNAcylation involvement in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2.

    PubMed

    Ding, Fang; Yu, Lu; Wang, Meihui; Xu, Shengjie; Xia, Qiang; Fu, Guosheng

    2013-08-01

    Continuous hyperglycemia is considered to be the most significant pathogenesis of diabetic cardiomyopathy, which manifests as cardiac hypertrophy and subsequent heart failure. O-GlcNAcylation has attracted attention as a post-translational protein modification in the past decade. The role of O-GlcNAcylation in high glucose-induced cardiomyocyte hypertrophy remains unclear. We studied the effect of O-GlcNAcylation on neonatal rat cardiomyocytes that were exposed to high glucose and myocardium in diabetic rats induced by streptozocin. High glucose (30 mM) incubation induced a greater than twofold increase in cell size and increased hypertrophy marker gene expression accompanied by elevated O-GlcNAcylation protein levels. High glucose increased ERK1/2 but not p38 MAPK or JNK activity, and cyclin D2 expression was also increased. PUGNAc, an inhibitor of β-N-acetylglucosaminidase, enhanced O-GlcNAcylation and imitated the effects of high glucose. OGT siRNA and ERK1/2 inhibition with PD98059 treatment blunted the hypertrophic response and cyclin D2 upregulation. OGT inhibition also prevented ERK1/2 activation. We also observed concentric hypertrophy and similar changes of O-GlcNAcylation level, ERK1/2 activation and cyclin D2 expression in myocardium of diabetic rats induced by streptozocin. In conclusion, O-GlcNAcylation plays a role in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2. PMID:23665912

  2. 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

  3. Let-7 modulates acquired resistance of ovarian cancer to Taxanes via IMP-1-mediated stabilization of MDR1

    PubMed Central

    Boyerinas, Benjamin; Park, Sun-Mi; Murmann, Andrea E.; Gwin, Katja; Montag, Anton G.; Zillardt, Marion R.; Hua, You-Jia; Lengyel, Ernst; Peter, Marcus E.

    2011-01-01

    Summary Ovarian cancer patients frequently develop resistance to chemotherapy regiments utilizing Taxol and carboplatin. One of the resistance factors that protects cancer cells from Taxol-based therapy is multi-drug resistance 1 (MDR1). micro(mi)RNAs are small noncoding RNAs that negatively regulate protein expression. Members of the let-7 family of miRNAs are downregulated in many human cancers, and low let-7 expression has been correlated with resistance to microtubule targeting drugs (Taxanes), although little is known that would explain this activity. We now provide evidence that, while let-7 is not a universal sensitizer of cancer cells to Taxanes, it affects acquired resistance of cells to this class of drugs by targeting IMP-1, resulting in de-stabilization of the mRNA of MDR1. Introducing let-7g into ADR-RES cells expressing both IMP-1 and MDR1 reduced expression of both proteins rendering the cells more sensitive to treatment with either Taxol or vinblastine without affecting the sensitivity of the cells to carboplatin, a non-MDR1 substrate. This effect could be reversed by reintroducing IMP-1 into let-7g high/MDR1 low cells causing MDR1 to again become stabilized. Consistently, many relapsed ovarian cancer patients tested before and after chemotherapy were found to downregulate let-7 and to co-upregulate IMP-1 and MDR1, and the increase in the expression levels of both proteins after chemotherapy negatively correlated with disease-free time before recurrence. Our data point at IMP-1 and MDR1 as indicators for response to therapy, and at IMP-1 as a novel therapeutic target for overcoming multidrug resistance of ovarian cancer. PMID:21618519

  4. Lipopolysaccharide inhibits transforming growth factor-beta1-stimulated Smad6 expression by inducing phosphorylation of the linker region of Smad3 through a TLR4-IRAK1-ERK1/2 pathway.

    PubMed

    Kim, Eun-Ye; Kim, Byung-Chul

    2011-03-01

    Smad6, one of the inhibitory Smads, plays an important role in transforming growth factor-beta1 (TGF-β1)-mediated negative regulation of pro-inflammatory signaling. In this study, we found that bacterial endotoxin lipopolysaccharide (LPS) inhibits TGF-β1-induced expression of Smad6 in RAW264.7 cells. This repression was accompanied by increased Smad3 linker phosphorylation at Thr-179 and Ser-208 and was dependent on ERK1/2 activity via the TLR4-IRAK1-linked signaling cascade. The expression of a mutant Smad3, that lacks the phosphorylation sites in the linker regions, significantly reversed the inhibitory effect of LPS on TGF-β1-induced Smad6 expression and its anti-inflammatory capacity. Collectively, our findings show how LPS pro-inflammatory signal antagonizes the anti-inflammatory activity of TGF-β1. PMID:21295571

  5. 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.

  6. Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway

    PubMed Central

    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

  7. 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

  8. 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

  9. 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. PMID:22820196

  10. 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

  11. 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

  12. 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.

  13. Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

    PubMed

    Lu, Ming-Chi; Lai, Ning-Sheng; Yin, Wen-Yao; Yu, Hui-Chun; Huang, Hsien-Bin; Tung, Chien-Hsueh; Huang, Kuang-Yung; Yu, Chia-Li

    2013-04-01

    In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α . PMID:23188524

  14. Activation of CaMKII and ERK1/2 contributes to the time-dependent potentiation of Ca2+ response elicited by repeated application of capsaicin in rat DRG neurons

    PubMed Central

    Daugherty, Stephanie L.; de Groat, William C.

    2011-01-01

    When capsaicin is applied repeatedly to dorsal root ganglion (DRG) neurons for brief periods (10–15 s) at short intervals (5–10 min), the evoked responses rapidly decline, a phenomenon termed tachyphylaxis. In addition to this phenomenon, the present study using Ca2+ imaging revealed that repeated application of capsaicin to rat dissociated DRG neurons at longer intervals (20–40 min) or during multiple applications at short intervals elicited an enhancement of the responses, termed potentiation. The potentiation occurred in 50–60% of the capsaicin-responsive cells, on average representing a 20- to 30% increase in the peak amplitude of the Ca2+ signal, and was maximal at a 40-min application interval. An analysis of the mechanisms underlying potentiation revealed that it was suppressed by block of Ca2+/calmodulin-dependent protein kinase II (CaMKII) with 5 μM KN-93 or block of the activation of extracellular signal-regulated kinase (ERK) 1/2 with 2 μM U-0126. Lowering the extracellular Ca2+ concentration from 2 to 1 mM or pretreatment with deltamethrin (1 μM), which blocks calcineurin and tachyphylaxis, enhanced potentiation. Potentiation was not affected by: 1) inhibition of protein kinase C or protein kinase A, 2) block of the three subtypes of neurokinin receptors, or 3) block of the trafficking of transient receptor potential V1 channel to the membrane. These results indicate that the potentiation is a slowly developing Ca2+-modulated process that is mediated by a complex intracellular signaling pathway involving activation of CaMKII and ERK1/2. Potentiation may be an important peripheral autosensitization mechanism that occurs independently of the pronociceptive effects of inflammatory mediators and neurotrophic factors. PMID:21178121

  15. 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

  16. Action of methyl-, propyl- and butylparaben on GPR30 gene and protein expression, cAMP levels and activation of ERK1/2 and PI3K/Akt signaling pathways in MCF-7 breast cancer cells and MCF-10A non-transformed breast epithelial cells.

    PubMed

    Wróbel, Anna Maria; Gregoraszczuk, Ewa Łucja

    2015-10-14

    In the present study, we examined cAMP levels and activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways in response to the actions of parabens on GPR30 in MCF-7 and MCF-10A cells. Cells were exposed to methyl-, propyl- or butylparaben at a concentration of 20nM; 17-β-estradiol (10nM) was used as a positive control. 17β-estradiol and all tested parabens increased GPR30 gene and protein expression in MCF-7 and MCF-10A cells. No parabens affected cAMP levels in either cell line, with the exception of propylparaben in MCF-10A cells. 17β-estradiol, propylparaben, and butylparaben increased phosphorylation of ERK1/2 in MCF-7 cells, whereas 17β-estradiol, methyl- and butylparaben, but not propylparaben, increased phosphorylation of ERK1/2 in MCF-10A cells. Akt activation was noted only in MCF-7 cells and only with propylparaben treatment. Collectively, the data presented here point to a nongenomic mechanism of action of parabens in activation GPR30 in both cancer and non-cancer breast cell lines through βγ dimer-mediated activation of the ERK1/2 pathway, but not the cAMP/PKA pathway. Moreover, among investigated parabens, propylparaben appears to inhibit apoptosis in cancer cells through activation of Akt kinases, confirming conclusions suggested by our previously published data. Nevertheless, continuing research on the carcinogenic action of parabens is warranted. PMID:26253279

  17. The ERK1/2 pathway participates in the upregulation of the expression of mesenteric artery α1 receptors by intravenous tail injections of mmLDL in mice.

    PubMed

    Wang, Jun-Jie; Chen, Xiao-Lan; Xu, Cang-Bao; Jiang, Gao-Feng; Lin, Jie; Liu, En-Qi; Qin, Xu-Ping; Li, Jie

    2016-02-01

    Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. However, no studies examining the effect of mmLDL on vascular smooth muscle receptors have been released. The current study investigated the effect of mmLDL on the mesenteric artery α1 adrenoceptor and the molecular mechanisms. Mice were divided into the normal saline (NS), mmLDL, and mmLDL+U0126 groups. In the mmLDL+U0126 group, the animals were subjected to an intravenous tail injection of mmLDL and an intraperitoneal injection of U0126. Vascular tension caused by noradrenaline (NA) in mesenteric arteries was measured with a sensitive myograph system. The serum levels of oxLDL, TNF-α, and IL-1β were detected using enzyme-linked immunosorbent assays. The expressions of the α1 adrenoceptor, the α2 adrenoceptor, TNF-α, IL-1β, and pERK1/2 were detected using real-time polymerase chain reactions and Western blot analysis. Compared with the NS group, the mmLDL group exhibited a noticeably enhanced NA shrinkage dose-response curve and a significantly increased Emax value (P<0.01). Prazosin (α1 adrenoceptor antagonist) caused a noticeable right shift of the dose-response curve. U0126 inhibited the increases in the serum levels and vessel wall expression of IL-1β and TNF-α and enhanced the NA shrinkage dose-response curve caused by mmLDL, as observed by a significantly decreased Emax value (P<0.01). It inhibited the increased α1 adrenoceptor expression caused by mmLDL. The serum levels of IL-1β and TNF-α demonstrated a positive correlation with the NA-induced maximum shrinkage percentage. U0126 inhibited the mmLDL-induced increase in the pERK1/2 protein level in the vessel wall. In conclusion, mmLDL increased the serum levels of IL-1β and TNF-α in vivo by activating the ERK1/2 pathway, which resulted in α1 receptor-mediated vasoconstriction and an increase in the expression of α1 adrenoceptor. The results of this study may provide new ideas for the prevention

  18. 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

  19. Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

    PubMed

    Blake, James F; Burkard, Michael; Chan, Jocelyn; Chen, Huifen; Chou, Kang-Jye; Diaz, Dolores; Dudley, Danette A; Gaudino, John J; Gould, Stephen E; Grina, Jonas; Hunsaker, Thomas; Liu, Lichuan; Martinson, Matthew; Moreno, David; Mueller, Lars; Orr, Christine; Pacheco, Patricia; Qin, Ann; Rasor, Kevin; Ren, Li; Robarge, Kirk; Shahidi-Latham, Sheerin; Stults, Jeffrey; Sullivan, Francis; Wang, Weiru; Yin, Jianping; Zhou, Aihe; Belvin, Marcia; Merchant, Mark; Moffat, John; Schwarz, Jacob B

    2016-06-23

    The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity. PMID:27227380

  20. Discovery and structure activity relationship study of novel indazole amide inhibitors for extracellular signal-regulated kinase1/2 (ERK1/2).

    PubMed

    Li, Lei; Liu, Feifei; Jin, Nan; Tang, Shuai; Chen, Zhuxi; Yang, Xiaotong; Ding, Jian; Geng, Meiyu; Jiang, Lei; Huang, Min; Cao, Jianhua

    2016-06-01

    The discovery and optimization of a series of indazole amide based extracellular signal-regulated kinase inhibitors via structure/knowledge based drug design and kinase screen is reported. The optimized compounds demonstrate potent inhibition of ERK1/2 enzyme activity, growth of BRAF mutant HT29 cells and ERK signaling in HT29 cells. PMID:27106711

  1. ERK1/2 Phosphorylation in the Rat Supraoptic Nucleus, Dorsal Raphe Nucleus, and Locus Coeruleus Neurons Following Noxious Stimulation to the Hind Paw.

    PubMed

    Donnerer, Josef; Liebmann, Ingrid

    2016-01-01

    Phospho-ERK1/2 (pERK1/2) fluorescence-immunohistochemistry is specifically well suited to mirror neuronal activity in the pain pathway at the cellular level. This study employed this method to visualize neuronal activity in 3 rat CNS nuclei following an acute noxious stimulation. The rat hind paw was stimulated either by heat or by a sequence of mustard oil and heat. Two min after the thermal stimulation or after the combined mustard oil and thermal stimulation, there was a significant increase in cells showing pERK1/2 immunoreactivity in the supraoptic nucleus (SON), in the dorsal raphe nucleus (DRN), and in the locus coeruleus (LC). Pretreatment with the opioid analgesic morphine or the N-methyl-D-aspartate antagonist MK-801 markedly attenuated ERK1/2 phosphorylation. These findings support the concept that the SON, the DRN, and the LC are integrated into pain processing at the hypothalamic and brain stem level. PMID:26599629

  2. 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

  3. Activation of EGFR/ERBB2 via Pathways Involving ERK1/2, P38 MAPK, AKT and FOXO Enhances Recovery of Diabetic Hearts from Ischemia-Reperfusion Injury

    PubMed Central

    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. PMID:22720029

  4. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

    SciTech Connect

    Ilowski, Maren; Putz, Christine; Weiss, Thomas S.; Brand, Stephan; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang Erwin

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  5. Glutathione, N-acetylcysteine and lipoic acid down-regulate starvation-induced apoptosis, RANKL/OPG ratio and sclerostin in osteocytes: involvement of JNK and ERK1/2 signalling.

    PubMed

    Fontani, Filippo; Marcucci, Gemma; Iantomasi, Teresa; Brandi, Maria Luisa; Vincenzini, Maria Teresa

    2015-04-01

    Osteocyte apoptosis due to microdamage and/or oxidative stress is related to increased local bone turnover and resorption observed in various bone diseases. Previous data on osteoblasts and osteoclasts have linked reactive oxygen species and antioxidants to bone remodelling. This study performs a comprehensive analysis on the effect of antioxidants such as glutathione (GSH), N-acetylcysteine and lipoic acid (LA) on starvation-induced osteocyte apoptosis and on cytokines involved in bone remodelling such as the receptor activator kB ligand (RANKL), osteoprotegerin (OPG) and sclerostin. For this study, apoptosis was induced by serum starvation in a murine osteocyte-like cell line MLO-Y4; this condition mimics in part osteocyte apoptosis due to microdamage. The results show that starvation-induced apoptosis and expression of RANKL, OPG and sclerostin are redox regulated processes. All antioxidants are able to inhibit the apoptosis due to starvation. They down-regulate the expression and the release of RANKL, the expression of sclerostin and RANKL/OPG ratio, whereas they only in part up-regulate OPG expression. Antioxidants mediate their effect on starvation-induced apoptosis by JNK signalling and on cytokine expression by both JNK and ERK1/2 activities. This study shows the possible involvement of biological antioxidants such as GSH and LA on redox regulated mechanisms related to apoptosis and expression of cytokines involved in bone remodelling. Moreover, it suggests that both JNK and ERK1/2 may be useful biological targets for drugs affecting bone diseases associated with increased oxidative stress. PMID:25660312

  6. 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

  7. Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling

    PubMed Central

    LI, WENKAI; WEI, SHENG; LIU, CHAOXU; SONG, MINGYU; WU, HUA; YANG, YONG

    2016-01-01

    An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process. PMID:26531757

  8. 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

  9. 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

  10. Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

    PubMed Central

    He, Jin; Li, Yan-Shu; Wang, Xiu-Mei; Ai, Hong-Jun; Cui, Fu-Zhai

    2013-01-01

    The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering. PMID:24069599

  11. 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

  12. Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

    SciTech Connect

    He Xiaoqing; Chen Rui; Yang Ping; Li Aiping; Zhou Jianwei; Liu Qizhan . E-mail: drqzliu@126.com

    2007-04-01

    Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 {mu}M) arsenite but inhibited at higher concentrations (5 and 10 {mu}M). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 {mu}M arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 {mu}M) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 {mu}M SP600125 or ERK1/2 by 100 {mu}M PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process.

  13. 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.

  14. 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. PMID:21596018

  15. Activation of ERK1/2 and p38 kinases by polycyclic aromatic hydrocarbons in rat liver epithelial cells is associated with induction of apoptosis

    SciTech Connect

    Andrysik, Zdenek; Machala, Miroslav; Chramostova, Katerina; Hofmanova, Jirina; Kozubik, Alois; Vondracek, Jan . E-mail: vondracek@ibp.cz

    2006-03-15

    Deregulation of various signaling pathways, linked either to induction of cell proliferation or to modulation of cellular differentiation and apoptosis, has been proposed to contribute to carcinogenicity of polycyclic aromatic hydrocarbons (PAHs). In the present study, we investigated effects of the PAHs previously shown to induce cell proliferation and/or apoptosis in contact-inhibited rat liver epithelial WB-F344 cells, with an aim to define the role of mitogen-activated protein kinases in both events. We found that only strong genotoxin dibenzo[a,l]pyrene (DBalP) activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase, but not c-Jun N-terminal kinases (JNKs), at concentrations inducing both apoptosis and phosphorylation of p53 tumor suppressor at serine 15 residue. In contrast, the PAHs stimulating cell proliferation in WB-F344 cell line had no effect on activation of ERK1/2, p38 or JNKs. Synthetic inhibitors of ERK1/2 activation (U0126) or p38 kinase activity (SB203580) prevented both apoptosis and induction of p53 phosphorylation by DBalP. Pifithrin-{alpha}, inhibitor of p53 transcriptional activity, prevented induction of apoptosis and activation of ERK1/2 and p38. Taken together, our data suggest that both ERK1/2 and p38 are activated in response to DBalP and that they might be involved in regulation of cellular response to DNA damage induced by DBalP, while neither kinase is involved in the release from contact inhibition induced by PAHs.

  16. Pro-oxidant/antioxidant balance controls pancreatic β-cell differentiation through the ERK1/2 pathway

    PubMed Central

    Hoarau, E; Chandra, V; Rustin, P; Scharfmann, R; Duvillie, B

    2014-01-01

    During embryogenesis, the intrauterine milieu affects cell proliferation, differentiation, and function by modifying gene expression in susceptible cells, such as the pancreatic β-cells. In this limited energy environment, mitochondrial dysfunction can lead to overproduction of reactive oxygen species (ROS) and to a decline in β-cell function. In opposition to this toxicity, ROS are also required for insulin secretion. Here we investigated the role of ROS in β-cell development. Surprisingly, decreasing ROS production in vivo reduced β-cell differentiation. Moreover, in cultures of pancreatic explants, progenitors were highly sensitive to ROS stimulation and responded by generating β-cells. ROS enhanced β-cell differentiation through modulation of ERK1/2 signaling. Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal β-cell differentiation. This study highlights the role of the redox balance on β-cell development and provides information that will be useful for improving β-cell production from embryonic stem cells, a step in cell therapy for diabetes. PMID:25341041

  17. M3 muscarinic receptors promote cell survival through activation of the extracellular regulated kinase (ERK1/2) pathway.

    PubMed

    Greenwood, Jeffrey M; Dragunow, Michael

    2010-08-25

    Activation of certain subtypes of the muscarinic acetylcholine receptor can enhance cell survival. In SK-N-SH human neuroblastoma cells, muscarinic acetylcholine receptor activation induces phosphorylation of CREB and induction of EGR1, transcription factors associated with cell growth and survival. We identified the M3 muscarinic acetylcholine receptor subtype as being primarily responsible for these transcription factor responses after stimulation with carbachol, using subtype-preferring receptor antagonists and muscarinic snake toxins. In a cell survival/death model in SK-N-SH cells deprived of serum growth factors, carbachol increased cell viability, an effect blocked by the non-specific muscarinic antagonist atropine and the M3-preferring antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), suggesting that the M3 receptor is also driving the survival response in these cells. This cytoprotection is largely dependent on activation of the p44/42 extracellular regulated kinase (ERK1/2) pathway. Understanding such survival signalling pathways is important for both potential interventions in neurodegenerative disease and for targeting neuroblastoma and malignancies of the central nervous system. PMID:20519144

  18. 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

  19. Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2.

    PubMed

    Kocić, Jelena; Santibañez, Juan F; Krstić, Aleksandra; Mojsilović, Slavko; Dorđević, Ivana Okić; Trivanović, Drenka; Ilić, Vesna; Bugarski, Diana

    2012-04-01

    The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment. PMID:22285818

  20. FasL expression in cardiomyocytes activates the ERK1/2 pathway, leading to dilated cardiomyopathy and advanced heart failure.

    PubMed

    Huby, Anne-Cecile; Turdi, Subat; James, Jeanne; Towbin, Jeffrey A; Purevjav, Enkhsaikhan

    2016-02-01

    Increase in the apoptotic molecule Fas ligand (FasL) in serum and cardiomyocytes has been shown to be associated with progressive dilated cardiomyopathy (DCM) and congestive heart failure (CHF) in humans. However, the underlying mechanism(s) of FasL-related deterioration of heart function remain obscure. The aim of the present study is to determine roles of myocardial FasL in the activation of alternative pathways such as extracellular-signal-regulated kinase 1/2 (ERK1/2), inflammation or fibrosis and to identify effective treatments of progressive DCM and advanced CHF. Transgenic mice with cardiomyocyte-specific overexpression of FasL were investigated and treated with an ERK1/2 inhibitor (U-0126), losartan (los), prednisolone (pred) or placebo. Morpho-histological and molecular studies were subsequently performed. FasL mice showed significantly higher mortality compared with wild-type (WT) littermates due to DCM and advanced CHF. Prominent perivascular and interstitial fibrosis, increased interleukin secretion and diffuse CD3-positive cell infiltration were evident in FasL hearts. Up-regulation of the short form of Fas-associated death domain (FADD)-like interleukin 1β-converting enzyme (FLICE) inhibitory protein (s-FLIP), RIP (receptor-interacting protein) and ERK1/2 and down-regulation of transforming growth factor beta 1 (TGFβ1) and nuclear factor-κB (NF-κB) was determined in the myocardium, whereas expression of ERK1/2, periostin (Postn) and osteopontin increased in cardiac fibroblasts. U-0126 and los increased CHF survival by 75% compared with pred and placebo groups. U-0126 had both anti-fibrotic and anti-apoptotic effects, whereas los reduced fibrosis only. Myocardial FasL expression in mice activates differential robust fibrotic, apoptotic and inflammatory responses via ERK1/2 in cardiomyocytes and cardiac fibroblasts inducing DCM and CHF. Blocking the ERK1/2 pathway prevented progression of FasL-induced DCM and CHF by reducing fibrosis, inflammation

  1. Quercetin suppresses inflammation by reducing ERK1/2 phosphorylation and NF kappa B activation in Leptin-induced Human Umbilical Vein Endothelial Cells (HUVECs)

    PubMed Central

    2013-01-01

    Background High concentrations of plasma leptin and the release of pro-inflammatory cytokines in leptin-resistance in obesity have been reported to trigger endothelial dysfunction. The objective of this study was to elucidate the role of quercetin in modulating leptin-induced inflammation as assessed by the levels of Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion in umbilical vein endothelial cells (HUVECs) in vitro. Findings HUVECs were exposed to either control levels (0 ng/ml) or 500 ng/mL leptin (L) for 48 hours, followed by control or 125 uM quercetin (Q) for another 6 h. The experimental groups were as follows: L0Q0, L0Q125, L500Q0, L500Q125. The presence of the short chain leptin receptor isoform Ob-Ra in HUVECs was determined by Western blot and immunocytochemistry analyses. Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion were quantified by ELISA, and NF-kappa B activationby immunofluorescence staining. Our results showed that Ob-Ra expression, ERK1/2 phosphorylation and NF-kappa B activation increased significantly after 500 ng/mL leptin exposure (1.8x, 1.5x, 6.2x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), but were reduced by addition of 125 uM quercetin (0.7x, 0.3x and 0.4x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), and that quercetin could also partially suppress leptin-induced TNF-alpha secretion (3.8x) by 0.8x. Conclusion Exposure of HUVECs to leptin up-regulated Ob-Ra expression and elevated ERK1/2 phosphorylation and NFkB activation, and increased TNF-alpha secretion. These effects strongly suppressed by quercetin, with the exception of TNF-alpha which was partially suppressed. The findings might be of clinical significance, as endothelial dysfunction that could lead to cardiovascular disease is preventable, and quercetin is a natural compound found in various plants and fruits. PMID:23856194

  2. N-Formyl peptides drive mitochondrial damage associated molecular pattern induced neutrophil activation through ERK1/2 and P38 MAP kinase signalling pathways.

    PubMed

    Hazeldine, Jon; Hampson, Peter; Opoku, Francis Adusei; Foster, Mark; Lord, Janet M

    2015-01-01

    Traumatic injury results in a systemic inflammatory response syndrome (SIRS), a phenomenon characterised by the release of pro-inflammatory cytokines into the circulation and immune cell activation. Released from necrotic cells as a result of tissue damage, damage associated molecular patterns (DAMPs) are thought to initiate the SIRS response by activating circulating immune cells through surface expressed pathogen recognition receptors. Neutrophils, the most abundant leucocyte in human circulation, are heavily implicated in the initial immune response to traumatic injury and have been shown to elicit a robust functional response to DAMP stimulation. Here, we confirm that mitochondrial DAMPs (mtDAMPs) are potent activators of human neutrophils and show for the first time that signalling through the mitogen-activated-protein-kinases p38 and extracellular-signal-related-kinase 1/2 (ERK1/2) is essential for this response. At 40 and/or 100 μg/ml, mtDAMPs activated human neutrophils, indicated by a significant reduction in the surface expression of L-selectin, and triggered a number of functional responses from both resting and tumour necrosis factor-α primed neutrophils, which included reactive oxygen species (ROS) generation, degranulation, secretion of interleukin-8 and activation of p38 and ERK1/2 MAPKs. Pre-treatment of neutrophils with Cyclosporin H, a selective inhibitor of formyl peptide receptor-1 (FPR-1), significantly inhibited mtDAMP-induced L-selectin shedding as well as p38 and ERK1/2 activation, suggesting that N-formyl peptides are the main constituents driving mtDAMP-induced neutrophil activation. Indeed, no evidence of L-selectin shedding or p38 and ERK1/2 activation was observed in neutrophils challenged with mitochondrial DNA alone. Interestingly, pharmacological inhibition of p38 or ERK1/2 either alone or in combination significantly inhibited L-selectin shedding and IL-8 secretion by mtDAMP-challenged neutrophils, revealing for the first time

  3. Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

    PubMed Central

    Menakongka, Apaporn; Suthiphongchai, Tuangporn

    2010-01-01

    AIM: To investigate the role of hepatocyte growth factor (HGF) in cholangiocarcinoma (CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS: Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines, HuCCA-1 and KKU-M213, using Transwell in vitro assay. Levels of proteins of interest and their phosphorylated forms were determined by Western blotting. Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS: Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line. HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization, but did not affect the levels of secreted matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator, key matrix degrading enzymes involved in cell invasion. Concomitantly, HGF stimulated Akt and extracellular signal-regulated kinase (ERK)1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines. Inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway by the PI3K inhibitor, LY294002, markedly suppressed HGF-stimulated invasion of both CCA cell lines, and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION: These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type. PMID:20135719

  4. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway

    PubMed Central

    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. PMID:25815516

  5. hesperidin induces paraptosis like cell death in hepatoblastoma, HepG2 Cells: involvement of ERK1/2 MAPK [corrected].

    PubMed

    Yumnam, Silvia; Park, Hyeon Soo; Kim, Mun Ki; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Lee, Won Sup; Kim, Eun Hee; Cho, Jae Hyeon; Shin, Sung Chul; Kim, Gon Sup

    2014-01-01

    Hesperidin, a natural flavonoid abundantly present in Citrus is known for its anti-cancer, anti-oxidant and anti-inflammatory properties. In this study we examined the effect of hesperidin on HepG2 cells. HepG2 cells treated with various concentration of hesperidin undergo a distinct type of programed cell death. Cytoplasmic vacuolization, mitochondria and endoplasmic reticulum swelling and uncondensed chromatin were observed in hesperidin treated cells. DNA electrophoresis show lack of DNA fragmentation and western blot analysis demonstrates lack of caspase activation and PARP cleavage. It was observed that hesperidin induced cell death is nonautophagic and also activate mitogen activated protein kinase ERK1/2. Taken together, the data indicate that hesperidin induces paraptosis like cell death in HepG2 cells with the activation of ERK1/2. Thus our finding suggests that hesperidin inducing paraptosis may offer an alternative tool in human liver carcinoma therapy. PMID:24977707

  6. 17beta-estradiol-induced activation of ERK1/2 through endogenous androgen receptor-estradiol receptor alpha-Src complex in human prostate cells.

    PubMed

    Chieffi, Paolo; Kisslinger, Annamaria; Sinisi, Antonio A; Abbondanza, Ciro; Tramontano, Donatella

    2003-09-01

    We examined the effect of estrogens on mitogen-activated protein kinase (MAPK) in EPN cells, a line of epithelial cells derived from human normal prostate. 17beta-estradiol (E2) caused a rapid and transient activation of MAPK (ERK1/2) within 5 min. This effect was counteracted by the anti-estrogen ICI 182-780 and by MEK inhibitor PD098059. The activation of ERK1/2 through 17beta-estradiol triggered simultaneous association of endogenous androgen receptor, estrogen receptor alpha and Src. In addition, E2 stimulated the proliferation of EPN cells, suggesting that the formation of the ternary complex and the consequent activation of ERKs are implicated in the mechanism regulating proliferation of epithelial prostate cells. PMID:12888920

  7. 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

  8. 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

  9. 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

  10. Suppression of protein kinase C theta contributes to enhanced myogenesis In vitro via IRS1 and ERK1/2 phosphorylation

    PubMed Central

    2013-01-01

    Background Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2. Results In this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation. Conclusion Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state. PMID

  11. 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

  12. Increased nuclear stiffness via FAK-ERK1/2 signaling is necessary for synthetic mechano-growth factor E peptide-induced tenocyte migration

    PubMed Central

    Zhang, Bingyu; Luo, Qing; Chen, Zhen; Shi, Yisong; Ju, Yang; Yang, Li; Song, Guanbin

    2016-01-01

    We have previously reported that a synthetic mechano-growth factor (MGF) C-terminal E-domain with 25 amino acids (MGF-C25E) promotes rat tenocyte migration through the FAK-ERK1/2 signaling pathway. However, the role of the nucleus in MGF-C25E-promoted tenocyte migration and the molecular mechanisms involved remain unclear. In this study, we demonstrate that MGF-C25E increases the Young’s modulus of tenocytes through the FAK-ERK1/2 signaling pathway. This increase is not accompanied by an obvious change in the expression of Lamin A/C but is accompanied by significant chromatin condensation, indicating that MGF-C25E-induced chromatin condensation may contribute to the increased nuclear stiffness. Moreover, DNA methylation is observed in MGF-C25E-treated tenocytes. Inhibition of DNA methylation suppresses the elevation in chromatin condensation, in nuclear stiffness, and in tenocyte migration induced by MGF-C25E. The inhibition of the focal adhesion kinase (FAK) or extracellular signal regulated kinase 1/2 (ERK1/2) signals represses MGF-C25E-promoted DNA methylation. It also abolishes chromatin condensation, nuclear stiffness, and cell migration. Taken together, our results suggest that MGF-C25E promotes tenocyte migration by increasing nuclear stiffness via the FAK-ERK1/2 signaling pathway. This provides strong evidence for the role of nuclear mechanics in tenocyte migration and new insight into the molecular mechanisms of MGF-promoted tenocyte migration. PMID:26742689

  13. 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.

  14. Overexpression of survivin via activation of ERK1/2, Akt, and NF-κB plays a central role in vincristine resistance in multiple myeloma cells.

    PubMed

    Tsubaki, Masanobu; Takeda, Tomoya; Ogawa, Naoki; Sakamoto, Kotaro; Shimaoka, Hirotaka; Fujita, Arisa; Itoh, Tatsuki; Imano, Motohiro; Ishizaka, Toshihiko; Satou, Takao; Nishida, Shozo

    2015-04-01

    The acquisition of anti-cancer drug resistance is a major limitation of chemotherapy for multiple myeloma (MM) and it is thus important to identify the mechanisms by which MM cells develop such drug resistance. In a previous study, we showed that multidrug resistance (MDR) involves the overexpression of MDR1 and survivin in vincristine-resistant RPMI8226/VCR cells. However, the underlying mechanism of MDR remains unclear. In this study, we investigated the mechanism of MDR in RPMI8226/VCR cells, and found that RPMI8226/VCR cells exhibit increased levels of activated ERK1/2, Akt, and NF-κB, while the levels of activated mTOR, p38MAPK, and JNK do not differ between RPMI8226/VCR cells and their vincristine-susceptible counterparts. In addition, the inhibition of ERK1/2, Akt, or NF-κB by inhibitors reversed the drug-resistance of RPMI8226/VCR cells via the suppression of survivin expression, but did not affect MDR1 expression; RNA silencing of survivin expression completely reversed vincristine resistance, while MDR1 silencing only weakly suppressed vincristine resistance in RPMI8226/VCR cells. These results indicate that enhanced survivin expression via the activation of ERK1/2, Akt, and NF-κB plays a critical role in vincristine resistance in RPMI8226/VCR cells. Our findings suggest that ERK1/2, Akt, and NF-κB inhibitors are potentially useful as anti-MDR agents for the treatment of vincristine-resistant MM. PMID:25726084

  15. BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Li, Xiaoyan; Lu, Wenju; Fu, Xin; Zhang, Yi; Yang, Kai; Zhong, Nanshan; Ran, Pixin

    2013-01-01

    Abnormal bone morphogenetic protein (BMP) signaling has been implicated in the pathogenesis of pulmonary hypertension. We previously found that BMP4 elevated basal intracellular Ca2+ ([Ca2+]i) concentrations in distal pulmonary arterial smooth muscle cells (PASMCs), attributable in large part to enhanced store-operated Ca2+ entry through store-operated Ca2+ channels (SOCCs). Moreover, BMP4 up-regulated the expression of canonical transient receptor potential (TRPC) proteins thought to compose SOCCs. The present study investigated the signaling pathways through which BMP4 regulates TRPC expression and basal [Ca2+]i in distal PASMCs. Real-time quantitative PCR was used for the measurement of mRNA, Western blotting was used for the measurement of protein, and fluorescent microscopic for [Ca2+]i was used to determine the involvement of p38 and extracellular regulated kinase (ERK)–1/2 mitogen-activated protein kinase (MAPK) signaling in BMP4–induced TRPC expression and the elevation of [Ca2+]i in PASMCs. We found that the treatment of BMP4 led to the activation of both p38 MAPK and ERK1/2 in rat distal PASMCs. The induction of TRPC1, TRPC4, and TRPC6 expression, and the increases of [Ca2+]i caused by BMP4 in distal PASMCs, were inhibited by treatment with either SB203580 (10 μM), the selective inhibitor for p38 activation, or the specific p38 small interfering RNA (siRNA). Similarly, those responses induced by BMP4 were also abolished by treatment with PD98059 (5 μM), the selective inhibitor of ERK1/2, or by the knockdown of ERK1/2 using its specific siRNA. These results indicate that BMP4 participates in the regulation of Ca2+ signaling in PASMCs by modulating TRPC channel expression via activating p38 and ERK1/2 MAPK pathways. PMID:23526217

  16. 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

  17. mGluR3 promotes proliferation of human embryonic cortical neural progenitor cells by activating ERK1/2 and JNK2 signaling pathway in vitro.

    PubMed

    Guo, J; Zhou, X; Chen, Y; Bai, M; Yang, X; Zhao, K; Hao, W; Wei, W; Zhang, Y

    2014-01-01

    Metabotropic glutamate receptors (mGluRs) regulate the proliferation and differentiation of neural progenitor cells (NPCs) in brain; however, the mechanisms remain unknown. In this study, we investigated the effect of mGluR3 on the proliferation of human embryonic neural progenitor cells (NPCs), the expression of cyclin D1 and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). The results showed that mGluR3 agonist N-Acetylaspartylglutamate (NAAG) increased the proliferation of NPCs by increasing cell activity, diameter of neurospheres and cell division. In addition, mGluR3 siRNA decreased the NPC proliferation. The protein expressions of cyclin D1 increased with NAAG treatment and decreased after siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal protein kinase (JNK) signaling pathways were involved in the proliferation of NPCs. NAAG increased phosphorylation of ERK1/2 and JNK2 levels, and meanwhile p-p38 level decreased; but p-ERK1/2 and p-JNK2 levels decreased after siRNA treatment, and p-p38 level increased. ERK1/2 inhibitor U0126 and JNK2 inhibitor SP600125 attenuated the increase of proliferation induced by NAAG. These findings demonstrated that mGluR3 promoted the proliferation of human embryonic cortical NPCs and increased cyclin D1 expression by activating ERK1/2 and JNK2 signaling pathways in vitro, suggesting that mGluR3 may be a target molecule for regulating NPC proliferation in brain development. PMID:25198581

  18. 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

  19. 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

  20. MEK-ERK1/2-dependent FLNA overexpression promotes abnormal dendritic patterning in tuberous sclerosis independent of mTOR.

    PubMed

    Zhang, Longbo; Bartley, Christopher M; Gong, Xuan; Hsieh, Lawrence S; Lin, Tiffany V; Feliciano, David M; Bordey, Angélique

    2014-10-01

    Abnormal dendritic complexity is a shared feature of many neurodevelopmental disorders associated with neurological defects. Here, we found that the actin-crosslinking protein filamin A (FLNA) is overexpressed in tuberous sclerosis complex (TSC) mice, a PI3K-mTOR model of neurodevelopmental disease that is associated with abnormal dendritic complexity. Both under- and overexpression of FLNA in wild-type neurons led to more complex dendritic arbors in vivo, suggesting that an optimal level of FLNA expression is required for normal dendritogenesis. In Tsc1(null) neurons, knocking down FLNA in vivo prevented dendritic abnormalities. Surprisingly, FLNA overexpression in Tsc1(null) neurons was dependent on MEK1/2 but not mTOR activity, despite both pathways being hyperactive. In addition, increasing MEK-ERK1/2 activity led to dendritic abnormalities via FLNA, and decreasing MEK-ERK1/2 signaling in Tsc1(null) neurons rescued dendritic defects. These data demonstrate that altered FLNA expression increases dendritic complexity and contributes to pathologic dendritic patterning in TSC in an mTOR-independent, ERK1/2-dependent manner. PMID:25277454

  1. MEK-ERK1/2-dependent FLNA overexpression promotes abnormal dendritic patterning in tuberous sclerosis independent of mTOR

    PubMed Central

    Zhang, Longbo; Bartley, Christopher M.; Gong, Xuan; Hsieh, Lawrence S.; Lin, Tiffany V.; Feliciano, David M.; Bordey, Angélique

    2014-01-01

    Summary Abnormal dendritic complexity is a shared feature of many neurodevelopmental disorders associated with neurological defects. Here, we found that the actin-crosslinking protein filamin A (FLNA) is overexpressed in tuberous sclerosis complex (TSC) mice, a PI3K-mTOR model of neurodevelopmental disease that is associated with abnormal dendritic complexity. Both under-and overexpression of FLNA in wild-type neurons led to more complex dendritic arbors in vivo, suggesting that an optimal level of FLNA expression is required for normal dendritogenesis. In Tsc1null neurons, knocking down FLNA in vivo prevented dendritic abnormalities. Surprisingly, FLNA overexpression in Tsc1null neurons was dependent on MEK1/2 but not mTOR activity despite both pathways being hyperactive. In addition, increasing MEK-ERK1/2 activity led to dendritic abnormalities via FLNA and decreasing MEK-ERK1/2 signaling in Tsc1null neurons rescued dendritic defects. These data demonstrate that altered FLNA expression increases dendritic complexity and contributes to pathologic dendritic patterning in TSC in an mTOR-independent, ERK1/2-dependent manner. PMID:25277454

  2. 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

  3. 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

  4. Cardiac concentric hypertrophy promoted by activated Met receptor is mitigated in vivo by inhibition of Erk1,2 signalling with Pimasertib.

    PubMed

    Sala, Valentina; Gallo, Simona; Gatti, Stefano; Medico, Enzo; Vigna, Elisa; Cantarella, Daniela; Fontani, Lara; Natale, Massimo; Cimino, James; Morello, Mara; Comoglio, Paolo Maria; Ponzetto, Antonio; Crepaldi, Tiziana

    2016-04-01

    Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies. PMID:26924269

  5. Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis via the PI3K/AKT and ERK1/2 MAPK Pathways.

    PubMed

    Lim, Whasun; Park, Sunwoo; Bazer, Fuller W; Song, Gwonhwa

    2016-12-01

    Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat, and sprouts. It has a variety of biological properties including anti-oxidant, anti-mutagenic, anti-carcinogenic, anti-inflammatory, anti-proliferative, and anti-spasmodic effects. Based on epidemiological and case-control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti-cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK, and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose-dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti-proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. J. Cell. Physiol. 231: 2690-2699, 2016. © 2016 Wiley Periodicals, Inc. PMID:26970256

  6. 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

  7. Cyclic Tensile Stress During Physiological Occlusal Force Enhances Osteogenic Differentiation of Human Periodontal Ligament Cells via ERK1/2-Elk1 MAPK Pathway

    PubMed Central

    Li, Lu; Han, Minxuan; Li, Sheng

    2013-01-01

    Physiological occlusal force constitutively exists in the oral environment and is important for periodontal homeostasis and remodeling. Cyclic tensile stress (CTS) triggers the biological response of periodontal ligament (PDL). However, a few reports have studied the correlation between CTS during physiological occlusal force and PDL cell activities such as osteogenic differentiation. In the present study, human PDL cells (hPDLCs) were subjected to 10% elongation CTS loading at 0.5 Hz for 24 h, which represents the physiological conditions of occlusal force. Gene expression microarray was used to investigate the mechano-induced differential gene profile and pathway analysis in vitro. The osteogenic relative factors, that is, SPP1, RUNX2, and SP7, were assessed by real-time PCR and Western blot. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was investigated by Western blot with a specific inhibitor. The expressions of SPP1, RUNX2, SP7, p-ERK1/2, and p-Elk1 were up-regulated after 10% CTS exposure. However, these up-regulated expressions were prevented by ERK1/2 inhibitor U0126 in the physiological occlusal force-applied hPDLCs. These results showed that 10% CTS could enhance osteogenic differentiation of hPDLCs via ERK1/2-Elk1 MAPK pathway, indicating that CTS during physiological occlusal force is a potent agent for PDL remodeling. PMID:23781879

  8. 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. PMID:24755484

  9. 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.

  10. 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

  11. 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. PMID

  12. 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

  13. Aspergillus fumigatus-induced Interleukin-8 Synthesis by Respiratory Epithelial Cells Is Controlled by the Phosphatidylinositol 3-Kinase, p38 MAPK, and ERK1/2 Pathways and Not by the Toll-like Receptor-MyD88 Pathway*

    PubMed Central

    Balloy, Viviane; Sallenave, Jean-Michel; Wu, Yongzheng; Touqui, Lhousseine; Latgé, Jean-Paul; Si-Tahar, Mustapha; Chignard, Michel

    2008-01-01

    Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-κB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-κB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis. PMID:18703508

  14. Aspergillus fumigatus-induced interleukin-8 synthesis by respiratory epithelial cells is controlled by the phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2 pathways and not by the toll-like receptor-MyD88 pathway.

    PubMed

    Balloy, Viviane; Sallenave, Jean-Michel; Wu, Yongzheng; Touqui, Lhousseine; Latgé, Jean-Paul; Si-Tahar, Mustapha; Chignard, Michel

    2008-11-01

    Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-kappaB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-kappaB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis. PMID:18703508

  15. 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

  16. MiR-494 is regulated by ERK1/2 and modulates TRAIL-induced apoptosis in non-small-cell lung cancer through BIM down-regulation.

    PubMed

    Romano, Giulia; Acunzo, Mario; Garofalo, Michela; Di Leva, Gianpiero; Cascione, Luciano; Zanca, Ciro; Bolon, Brad; Condorelli, Gerolama; Croce, Carlo M

    2012-10-01

    MicroRNAs (miRNAs) have an important role in the development of chemosensitivity or chemoresistance in different types of cancer. Activation of the ERK1/2 pathway is a major determinant of diverse cellular processes and cancer development and is responsible for the transcription of several important miRNAs. Here we show a link between the ERK1/2 pathway and BIM expression through miR-494. We blocked ERK1/2 nuclear activity through the overexpression of an ERK1/2 natural interactor, the protein PED/PEA15, and we performed a microRNA expression profile. miR-494 was the most down-regulated microRNA after ERK1/2 inactivation. Moreover, we found that miR-494 induced Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance in non-small-cell lung cancer (NSCLC) through the down-modulation of BIM. Elucidation of this undiscovered ERK1/2 pathway that regulates apoptosis and cell proliferation through miR-494 in NSCLC will greatly enhance our understanding of the mechanisms responsible for TRAIL resistance and will provide an additional arm for the development of anticancer therapies. PMID:23012423

  17. 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

  18. 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

  19. 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.

  20. GABA(A) Receptor Pi (GABRP) Stimulates Basal-like Breast Cancer Cell Migration through Activation of Extracellular-regulated Kinase 1/2 (ERK1/2)*

    PubMed Central

    Sizemore, Gina M.; Sizemore, Steven T.; Seachrist, Darcie D.; Keri, Ruth A.

    2014-01-01

    Breast cancer is a heterogeneous disease comprised of distinct subtypes predictive of patient outcome. Tumors of the basal-like subtype have a poor prognosis due to inherent aggressiveness and the lack of targeted therapeutics. Basal-like tumors typically lack estrogen receptor-α, progesterone receptor and HER2/ERBB2, or in other words they are triple negative (TN). Continued evaluation of basal-like breast cancer (BLBC) biology is essential to identify novel therapeutic targets. Expression of the pi subunit of the GABA(A) receptor (GABRP) is associated with the BLBC/TN subtype, and herein, we reveal its expression also correlates with metastases to the brain and poorer patient outcome. GABRP expression in breast cancer cell lines also demonstrates a significant correlation with the basal-like subtype suggesting that GABRP functions in the initiation and/or progression of basal-like tumors. To address this postulate, we stably silenced GABRP in two BLBC cell lines, HCC1187 and HCC70 cells. Decreased GABRP reduces in vitro tumorigenic potential and migration concurrent with alterations in the cytoskeleton, specifically diminished cellular protrusions and expression of the BLBC-associated cytokeratins, KRT5, KRT6B, KRT14, and KRT17. Silencing GABRP also decreases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) in both cell lines and selective inhibition of ERK1/2 similarly decreases the basal-like cytokeratins as well as migration. Combined, these data reveal a GABRP-ERK1/2-cytokeratin axis that maintains the migratory phenotype of basal-like breast cancer. GABRP is a component of a cell surface receptor, thus, these findings suggest that targeting this new signaling axis may have therapeutic potential in BLBC. PMID:25012653

  1. Carbocisteine attenuates TNF-α-induced inflammation in human alveolar epithelial cells in vitro through suppressing NF-κB and ERK1/2 MAPK signaling pathways

    PubMed Central

    Wang, Wei; Guan, Wei-jie; Huang, Rong-quan; Xie, Yan-qing; Zheng, Jin-ping; Zhu, Shao-xuan; Chen, Mao; Zhong, Nan-shan

    2016-01-01

    Aim: We previously proven that carbocisteine, a conventional mucolytic drug, remarkably reduced the rate of acute exacerbations and improved the quality of life in the patients with chronic obstructive pulmonary disease. In this study we investigated the mechanisms underlying the anti-inflammatory effects of carbocisteine in human alveolar epithelial cells in vitro. Methods: Human lung adenocarcinoma cell line A549 was treated with TNF-α (10 ng/mL). Carbocisteine was administered either 24 h prior to or after TNF-α exposure. The cytokine release and expression were measured using ELISA and qRT-PCR. Activation of NF-κB was analyzed with Western blotting, immunofluorescence assay and luciferase reporter gene assay. The expression of ERK1/2 MAPK signaling proteins was assessed with Western blotting. Results: Carbocisteine (10, 100, 1000 μmol/L), administered either before or after TNF-α exposure, dose-dependently suppressed TNF-α-induced inflammation in A549 cells, as evidenced by diminished release of IL-6 and IL-8, and diminished mRNA expression of IL-6, IL-8, TNF-α, MCP-1 and MIP-1β. Furthermore, pretreatment with carbocisteine significantly decreased TNF-α-induced phosphorylation of NF-κB p65 and ERK1/2 MAPK, and inhibited the nuclear translocation of p65 subunit in A549 cells. In an NF-κB luciferase reporter system, pretreatment with carbocisteine dose-dependently inhibited TNF-α-induced transcriptional activity of NF-κB. Conclusion: Carbocisteine effectively suppresses TNF-α-induced inflammation in A549 cells via suppressing NF-κB and ERK1/2 MAPK signaling pathways. PMID:26997568

  2. Paeonol enhances thrombus recanalization by inducing vascular endothelial growth factor 165 via ERK1/2 MAPK signaling pathway.

    PubMed

    Ye, Songshan; Liu, Xianjuan; Mao, Bingyu; Yang, Lei; Liu, Nuan

    2016-06-01

    Paeonol (2'-hydroxy-4'-methoxyacetophenone) is the major active compound of Mautan cortex and has been demonstrated to inhibit platelet aggregation in previous studies. The current study aimed to elucidate the underlying molecular mechanism of paeonol in recanalizing thrombi. The presence of indicators of prothrombotic state (PTS) in the serum of the model animals were determined by enzyme‑linked immunosorbent assay (ELISA) assay and the cytotoxicity of paeonol on human umbilical vein endothelial cell (HUVEC) cultures was estimated by 3‑(4,5 dimethylthiazol‑2‑yl)-2,5-diphenyltetrazolium bromide assay. The possible underlying signaling pathway involved in the interaction between paeonol and vascular endothelial growth factor 165 (VEGF165) was investigated using western blotting. The levels of 6‑keto‑prostaglandin F1α, fibronectin, and VEGF165 in serum were significantly upregulated by the treatment of paeonol while the levels of fibrinogen, D‑dimer, and thromboxane B2 were significantly downregulated (P<0.05). With increased paeonol concentration, the cell viability of HUVECs gradually decreased. The results of the western blot analysis demonstrated that paeonol increased the expression levels of phosphorylated‑extracellular signal‑regulated kinase (ERK1/2) and VEGF165 but had no marked effect on the expression level of ERK1/2. Paeonol has the potential to improve PTS and recanalize thrombi in animal models, which may be by the upregulation of VEGF165 via the ERK1/2 mitogen activated protein kinase signaling pathway. However, this positive effect depended on the concentration of paeonol used, an unsuitably high concentration of the compound exerted negative effects on the anti‑thrombosis signaling pathways. PMID:27082415

  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. 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

  5. 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

  6. ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer

    PubMed Central

    Yoshida, Takeshi; Song, Lanxi; Bai, Yun; Kinose, Fumi; Li, Jiannong; Ohaegbulam, Kim C.; Muñoz-Antonia, Teresita; Qu, Xiaotao; Eschrich, Steven; Uramoto, Hidetaka; Tanaka, Fumihiro; Nasarre, Patrick; Gemmill, Robert M.; Roche, Joëlle; Drabkin, Harry A.; Haura, Eric B.

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors. PMID:26789630

  7. 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

  8. Differential phosphorylation of translation initiation regulators 4EBP1, S6k1, and Erk 1/2 following inhibition of alcohol metabolism in mouse heart.

    PubMed

    Vary, Thomas C; Lang, Charles H

    2008-03-01

    Acute alcohol intoxication leads to an inhibition of protein synthesis in heart that results in part through altered phosphorylation of protein factors controlling mRNA translation initiation. The purpose of the present set of experiments was designed to examine the effects of inhibitors of ethanol metabolism on the phosphorylation of 4E-binding protein (4EBP1) and S6k1(Thr(389)), two factors regulating mRNA translation initiation. Phosphorylation of 4E-BP1, S6k1(Thr(389)), and Erk 1/2 was reduced 2 h following IP injection of alcohol. Pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH), did not attenuate the ethanol-induced decrease in phosphorylation of 4EBP1 and S6k1(Thr(389)). In contrast, 4-MP prevented the decrease in Erk 1/2 phosphorylation observed with acute ethanol intoxication. Pretreatment with cyanamide, an inhibitor of aldehyde dehydrogenase, did not attenuate the ethanol-induced decrease in phosphorylation S6k1(Thr(389)), but partially prevented the ethanol-induced lowering of 4EBP1 phosphorylation. The studies indicate that modulation of ethanol metabolism through inhibition of ADH or aldehyde dehydrogenase leads to preferential modulation of the phosphorylation of distinct myocardial signaling systems involved in regulating protein synthesis. PMID:18317950

  9. 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

  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

    2014-01-01

    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

  11. Loss of expressions of Dusp6, Sprouty4, and Sef, negative regulators of FGF2/ERK1/2 signaling, in the endometrium of women with adenomyosis.

    PubMed

    Guo, Qiufen; Zhang, Hui; Zhao, Xingbo; Fu, Yibing; Zhang, Jie; Li, Mingjiang

    2014-05-01

    Dual-specificity phosphatase 6 (Dusp6), Sprouty4, and similar expression to FGF (Sef) are negative modulators of FGF2/ERK1/2 signaling. The objective of the study was to evaluate the expressions of Dusp6, Sprouty4, and Sef in eutopic endometria of patients with adenomyosis. Endometria from 30 women with adenomyosis and 29 women without adenomyosis were used in this study. The expressions of Dusp6, Sprouty4, and Sef were investigated by immunohistochemical analysis. We found that Dusp6, Sprouty4, and Sef expressions were present in endometrial epithelial cells of normal endometria and eutopic endometria of adenomyosis. Weak immunostainings were noted in stromal cells in both endometria. No cyclical change was noted either in normal endometria or in eutopic endometria of adenomyosis during menstrual cycle. By immunohistochemical analysis, we found that eutopic endometria of adenomyosis showed significantly decreased Dusp6, Sprouty4, and Sef expressions compared with normal endometria. By in situ hybridization analysis, we found that the mRNA expressions of Dusp6, Sprouty4, and Sef were downregulated in eutopic endometria of adenomyosis compared with normal endometria. We conclude that downregulation of Dusp6, Sprouty4, and Sef--negative modulators of FGF2/ERK1/2 signaling--was present in eutopic endometria of adenomyosis, which may play critical roles in the development of adenomyosis. PMID:24681741

  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. 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

  14. STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease

    PubMed Central

    Lu, Sydney X.; Alpdogan, Onder; Lin, Janine; Balderas, Robert; Campos-Gonzalez, Roberto; Wang, Xiao; Gao, Guo-Jian; Suh, David; King, Christopher; Chow, Melanie; Smith, Odette M.; Hubbard, Vanessa M.; Bautista, Johanne L.; Cabrera-Perez, Javier; Zakrzewski, Johannes L.; Kochman, Adam A.; Chow, Andrew; Altan-Bonnet, Gregoire

    2008-01-01

    Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets.1–7 This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phospho-specific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD. PMID:18838616

  15. Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2

    PubMed Central

    Wurm, Stefanie; Zhang, Jisheng; Guinea-Viniegra, Juan; García, Fernando; Muñoz, Javier; Bakiri, Latifa; Ezhkova, Elena

    2015-01-01

    Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. PMID:25547114

  16. Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2.

    PubMed

    Wurm, Stefanie; Zhang, Jisheng; Guinea-Viniegra, Juan; García, Fernando; Muñoz, Javier; Bakiri, Latifa; Ezhkova, Elena; Wagner, Erwin F

    2015-01-15

    Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. PMID:25547114

  17. Traditional Korean Herbal Formula Samsoeum Attenuates Adipogenesis by Regulating the Phosphorylation of ERK1/2 in 3T3-L1 Cells

    PubMed Central

    Jeong, Soo-Jin; Yoo, Sae-Rom; Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Adipogenesis is the cell differentiation process from preadipocytes into adipocytes and the critical action in the development of obesity. In the present study, we conducted in vitro analyses to investigate the inhibitory effects of Samsoeum (SSE), a traditional herbal decoction. SSE had no significant cytotoxic effect against either the undifferentiated or differentiated 3T3-L1 cells. Oil Red O staining results showed that SSE significantly inhibited fat accumulation in adipocytes. SSE treatment consistently reduced the intracellular triglyceride content in the cells. SSE significantly inactivated glycerol-3-phosphate dehydrogenase (GPDH), a major link between carbohydrate and lipid metabolisms in 3T3-L1 adipocytes, and markedly inhibited the production of leptin, an important adipokine, in differentiated cells. SSE markedly suppressed the mRNA expression of the adipogenesis-related genes peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer binding protein-alpha (C/EBP-α), fatty acid synthase (FAS), lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4). Importantly, SSE increased the phosphorylation of ERK1/2, but not p38 MAPK and JNK, in adipose cells. Overall, our results indicate that SSE exerts antiadipogenic activity and modulates expressions of adipogenesis-related genes and ERK1/2 activation in adipocytes. PMID:26483846

  18. Sphingosine-1-Phosphate Induces the Migration of Thyroid Follicular Carcinoma Cells through the MicroRNA-17/PTK6/ERK1/2 Pathway

    PubMed Central

    Zhao, Shitao; Li, Jincheng

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid known to play a role in tumorigenesis and cancer progression. However, the molecular mechanisms of S1P regulated migration of papillary thyroid cancer cells are still unknown. In this study, we showed that S1P induced PTK6 mRNA and protein expression in two thyroid follicular cancer cell lines (ML-1 and FTC-133). Further studies demonstrated that induced PTK6 and its downstream signal component (ERK1/2) are involved in S1P-induced migration. Upon investigating the mechanisms behind this event, we found that miR-17 inhibited the expression of PTK6 through direct binding to its 3’-UTR. Through overexpression and knockdown studies, we found that miR-17 can significantly inhibit S1P-induced migration in thyroid follicular cancer cells. Interestingly, overexpression or knockdown of PTK6 or ERK1/2 effectively removed the inhibition of S1P-induced migration by miR-17. Furthermore, we showed that S1P decreased miR-17 expression levels. Meanwhile, in papillary thyroid cancers, miR-17 is downregulated and negatively associated with clinical staging, whereas PTK6 is upregulated and positively associated with clinical stages. Collectively, our work defines a novel signaling pathway implicated in the control of thyroid cancer migration. PMID:25748447

  19. 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.

  20. Mechanical Strain Promotes Osteogenesis of BMSCs from Ovariectomized Rats via the ERK1/2 but not p38 or JNK-MAPK Signaling Pathways.

    PubMed

    Zhang, P; Dai, Q; Ouyang, N; Yang, X; Wang, J; Zhou, S; He, N; Fang, B; Jiang, L

    2015-01-01

    Osteoporosis has become a world-wide health problem. As a promising intervention, mechanical strain is considered to be an important factor in bone remodeling. However, the underlying mechanisms are still not clarified clearly. In the present study, we aim to investigate the possible mechanism by which mechanical stimulation induces osteogenic differentiation of bone mesenchymal stem cells (BMSCs) from ovariectomized rats (OVX BMSCs). The results demonstrated that intermittent mechanical strain (IMS) promoted osteogenic differentiation of OVX BMSCs by activating Runt-related transcription factor 2 (Runx2). When the extracellular regulated kinase1/2-mitogen activated protein kinases (ERK1/2-MAPK) signaling pathway was blocked, the osteogenenic effects of IMS were diminished; while blocking of the p38-MAPK signaling pathway had little effect on subsequent osteogenic events. In addition, the phosphorylation level of JNK was not affected by IMS. Our results indicated that strain-induced osteogenic differentiation of OVX BMSCs may take effect via ERK1/2-MAPK not p38 or c-Jun N-terminal (JNK)-MAPK signaling pathway. These findings may have implications for physical treatment of osteoporosis in vitro. PMID:26299771

  1. (+)-2-(1-Hydroxyl-4-oxocyclohexyl) ethyl caffeate suppresses solar UV-induced skin carcinogenesis by targeting PI3-K, ERK1/2 and p38

    PubMed Central

    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

    2014-01-01

    For decades, skin cancer incidence has increased, mainly due to 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 HOEC, [(+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate], isolated from Incarvillea mairei var. grandiflora (Wehrhahn) Grierson. HOEC strongly inhibited neoplastic transformation of JB6 C14l cells without toxicity. PI3-K, 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 PI3-K, ERK1/2 and p38 kinase activities resulted in suppression of their downstream signaling pathways and AP-1 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 as a potent chemopreventive compound against skin carcinogenesis caused by solar UV exposure. PMID:24845061

  2. The Pro-Inflammatory Cytokine IL-22 Up-Regulates Keratin 17 Expression in Keratinocytes via STAT3 and ERK1/2

    PubMed Central

    Shi, Xiaowei; Jin, Liang; Feng, Zhenzhen; Hu, Lei; Wu, Yan; Wang, Gang

    2012-01-01

    Background To investigate the regulation of K17 expression by the pro-inflammatory cytokine IL-22 in keratinocytes and its important role in our previously hypothesized “K17/T cell/cytokine autoimmune loop” in psoriasis. Materials and Methods K17 expression was examined in the IL-22-treated keratinocytes by real-time quantitative PCR, ELISA, Western blot and Immunofluorescence. In addition, the signaling pathways involved in K17 regulation were investigated with related inhibitors and siRNAs. In addition, K17 expression was examined in the epidermis of IL-22-injected mouse skin. Results IL-22-induced K17 expression was confirmed in keratinocytes and the epidermis of IL-22-injected mouse skin at both mRNA and protein levels, which is an important complement to the autoimmune loop. We further investigated the regulatory mechanisms and found that both STAT3 and ERK1/2 were involved in the up-regulation of K17 expression induced by IL-22. Conclusion IL-22 up-regulates K17 expression in keratinocytes in a dose-dependent manner through STAT3- and ERK1/2-dependent mechanisms. These findings indicated that IL-22 was also involved in the K17/T cell/cytokine autoimmune loop and may play an important role in the progression of psoriasis. PMID:22808266

  3. 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

    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

  4. 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

  5. Myricetin inhibits advanced glycation end product (AGE)-induced migration of retinal pericytes through phosphorylation of ERK1/2, FAK-1, and paxillin in vitro and in vivo.

    PubMed

    Kim, Young Sook; Kim, Junghyun; Kim, Ki Mo; Jung, Dong Ho; Choi, Sojin; Kim, Chan-Sik; Kim, Jin Sook

    2015-02-15

    Advanced glycation end products (AGE) have been implicated in the development of diabetic retinopathy. Characterization of the early stages of diabetic retinopathy is retinal pericytes loss, which is the result of pericytes migration. In this study, we investigated the pathological mechanisms of AGE on the migration of retinal pericytes and confirmed the inhibitory effect of myricetin on migration in vitro and in vivo. Migration assays of bovine retinal pericytes (BRP) were induced using AGE-BSA and phosphorylation of Src, ERK1/2, focal adhesion kinase (FAK-1) and paxillin were determined using immunoblot analysis. Sprague-Dawley rats (6 weeks old) were injected intravitreally with AGE-BSA and morphological and immunohistochemical analysis of p-FAK-1 and p-paxillin were performed in the rat retina. Immunoblot analysis and siRNA transfection were used to study the molecular mechanism of myricetin on BRP migration. AGE-BSA increased BRP migration in a dose-dependent manner via receptor for AGEs (RAGE)-dependent activation of the Src kinase-ERK1/2 pathway. AGE-BSA-induced migration was inhibited by an ERK1/2 specific inhibitor (PD98059), but not by p38 and Jun N-terminal kinase inhibitors. AGE-BSA increased FAK-1 and paxillin phosphorylation in a dose- and time-dependent manner. These increases were attenuated by PD98059 and ERK1/2 siRNA. Phosphorylation of FAK-1 and paxillin was increased in response to AGE-BSA-induced migration of rat retinal pericytes. Myricetin strongly inhibited ERK1/2 phosphorylation and significantly suppressed pericytes migration in AGE-BSA-injected rats. Our results demonstrate that AGE-BSA participated in the pathophysiology of retinal pericytes migration likely through the RAGE-Src-ERK1/2-FAK-1-paxillin signaling pathway. Furthermore, myricetin suppressed phosphorylation of ERK 1/2-FAK-1-paxillin and inhibited pericytes migration. PMID:25450667

  6. Acquired thrombotic thrombocytopenic purpura due to antibody-mediated ADAMTS13 deficiency precipitated by a localized Castleman's disease: a case report.

    PubMed

    Benevides, Thais Celi Lopes; Orsi, Fernanda Andrade; Colella, Marina Pereira; Percout, Priscila de Oliveira; Moura, Muriel Silva; Dias, Maria Almeida; Lins, Betina Diniz; Paula, Erich Vinicius de; Vassallo, Jose; Annichino-Bizzachi, Joyce

    2015-01-01

    Acquired ADAMTS13 inhibitor causing thrombotic thrombocytopenic purpura (TTP) may be precipitated by some infections, inflammatory diseases or neoplasia. We reported a case of refractory TTP precipitated by a newly diagnosed localized Castleman's disease (CD). TTP was initially treated with plasma exchange and immunosuppressive therapy with corticosteroids; however the treatment failed to promote sustained response. During hospitalization, an abdominal tumor was diagnosed and resected; the histological analysis revealed a CD of hyaline-vascular variant rich stroma. After tumor removal, the patient achieved a long-lasting clinical remission and normalized ADAMTS13 activity. This clinical case describes a novel association of acquired ADAMTS13 inhibitor and CD. The antibody to ADAMTS13 developed along with the systemic manifestation of CD and promptly disappeared after the resection of the tumor. There are reports of neoplasia-associated thrombotic microangiopathy however direct evidence of CD-dependent ADAMTS13 inhibitor had not yet been reported. PMID:24853254

  7. 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

  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

    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

  9. 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.

  10. Relaxin Protects Rat Lungs from Ischemia-Reperfusion Injury via Inducible NO Synthase: Role of ERK-1/2, PI3K, and Forkhead Transcription Factor FKHRL1

    PubMed Central

    Alexiou, Konstantin; Wilbring, Manuel; Matschke, Klaus; Dschietzig, Thomas

    2013-01-01

    Introduction Early allograft dysfunction following lung transplantation is mainly an ischemia/reperfusion (IR) injury. We showed that relaxin-2 (relaxin) exerts a protective effect in lung IR, attributable to decreases in endothelin-1 (ET-1) production, leukocyte recruitment, and free radical generation. Here, we summarize our investigations into relaxin’s signalling. Materials and Methods Isolated rat lungs were perfused with vehicle or 5 nM relaxin (n = 6–10 each). Thereafter, experiments were conducted in the presence of relaxin plus vehicle, the protein kinase A inhibitors H-89 and KT-5720, the NO synthase (NOS) inhibitor L-NAME, the iNOS inhibitor 1400W, the nNOS inhibitor SMTC, the extracellular signal-regulated kinase-1/2 (ERK-1/2) inhibitor PD-98059, the phosphatidylinositol-3 kinase (PI3K) inhibitor wortmannin, the endothelin type-B (ETB) antagonist A-192621, or the glucocorticoid receptor (GR) antagonist RU-486. After 90 min ischemia and 90 min reperfusion we determined wet-to-dry (W/D) weight ratio, mean pulmonary arterial pressure (MPAP), vascular release of ET-1, neutrophil elastase (NE), myeloperoxidase (MPO), and malondialdehyde (MDA). Primary rat pulmonary vascular cells were similarly treated. Results IR lungs displayed significantly elevated W/D ratios, MPAP, as well as ET-1, NE, MDA, and MPO. In the presence of relaxin, all of these parameters were markedly improved. This protective effect was completely abolished by L-NAME, 1400W, PD-98059, and wortmannin whereas neither PKA and nNOS inhibition nor ETB and GR antagonism were effective. Analysis of NOS gene expression and activity revealed that the relaxin-induced early and moderate iNOS stimulation is ERK-1/2-dependent and counter-balanced by PI3K. Relaxin-PI3K-related phosphorylation of a forkhead transcription factor, FKHRL1, paralleled this regulation. In pulmonary endothelial and smooth muscle cells, FKHRL1 was essential to relaxin-PI3K signalling towards iNOS. Conclusion In this

  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-ERK1/2/ERK1/2 ratio was reduced (33%) in antidepressant-free when compared to antidepressant-treated MD subjects. The neurochemical adaptations of brain FADD (increased p-FADD and pro-survival p-FADD/FADD ratio), as well as its interaction with PEA-15, could play a major role to counteract the known activation of the mitochondrial apoptotic pathway in MD. PMID:25075716

  12. Sauchinone augments cardiomyocyte viability by enhancing autophagy proteins -PI3K, ERK(1/2), AMPK and Beclin-1 during early ischemia-reperfusion injury in vitro

    PubMed Central

    Thapalia, Bisharad Anil; Zhou, Zhen; Lin, Xianhe

    2016-01-01

    Background. Sauchinone has proved its anti-oxidant and anti-inflammatory properties in various animal tissues. This study sought to illustrate its regulatory nature on autophagy associated proteins (PI3K, ERK1/2, AMPK, and Beclin-1) during early cardiomyocyte ischemia and subsequent reperfusion. Methods. Cultured cardiomyocytes were subjected to simulated Ischemia/reperfusion with and without Sauchinone pretreatment and also in the presence of autophagy inhibitor (3-MA). Colorimetric analysis of CCK-8, LDH antibody assay as well as Western blot analysis were performed to observe the expressions of LC3B (II) and Beclin-1 protein (markers of autophagy), autophagy proteins (PI3K, ERK1/2 and AMPK) and apoptotic proteins (Bax and Bcl-2) and the results were quantified into their grey values and subjected to statistical analysis. Results. Sauchinone demonstrated cell survival enhancing properties with increase in CCK-8 (SD = 0.553±0.012) and decrease in LDH (SD = 0.183±0.054) expressions, both of which were best observed at test dose of 20 µmol/L. At this dose, there was increment in cellular autophagy as demonstrated by peaking of autophagy markers LC3B-II (p<0.05) and Beclin-1 (p<0.05) with strong correlations (r = 0.99). Similarly, the autophagy proteins, compared to control and I/R model, also showed a significant increased level with PI3K (p<0.0001), total p-ERK1/2 (p<0.0001) and p-AMPKα (p<0.0001). Simultaneously, a decrease in expressions of pro-apoptotic molecules Bax (r = 0.989, p<0.0001) with increment of in the anti-apoptotic protein Bcl-2 (r = 0.996, p<0.0001) was observed. The observed effects on cell density, viability and autophagy was abrogated in presence of 3-MA. Conclusions. Sauchinone enhances cell survival by promoting autophagy and inhibiting apoptosis in cardiomyocytes during early stages of Ischemia/reperfusion injury. PMID:27508047

  13. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

    SciTech Connect

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effects of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing

  14. Curcumin mediates oxaliplatin-acquired resistance reversion in colorectal cancer cell lines through modulation of CXC-Chemokine/NF-κB signalling pathway

    PubMed Central

    Ruiz de Porras, Vicenç; Bystrup, Sara; Martínez-Cardús, Anna; Pluvinet, Raquel; Sumoy, Lauro; Howells, Lynne; James, Mark I.; Iwuji, Chinenye; Manzano, José Luis; Layos, Laura; Bugés, Cristina; Abad, Albert; Martínez-Balibrea, Eva

    2016-01-01

    Resistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-κB signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-κB was hyperactivated in in vitro models of OXA-acq