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Sample records for induced map kinase

  1. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    PubMed

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  2. 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 ...

  3. Dependence of Mos-induced Cdc2 activation on MAP kinase function in a cell-free system.

    PubMed Central

    Huang, C Y; Ferrell, J E

    1996-01-01

    The progression of G2-arrested Xenopus laevis oocytes into meiotic M-phase is accompanied by the nearly simultaneous activation of p42 MAP kinase and Cdc2/cyclin B. This timing raises the possibility that the activation of one kinase might depend upon the other. Here we have examined whether Cdc2 activation requires p42 MAP kinase function. We have reconstituted Mos-induced Cdc2 activation in cell-free Xenopus oocyte extracts, and have found that Mos-induced Cdc2 activation requires active p42 MAP kinase, is inhibited by a MAP kinase phosphatase and is independent of protein synthesis. These findings indicate that p42 MAP kinase is an essential component of the M phase trigger in this system. Images PMID:8641282

  4. Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

    PubMed Central

    Giordano, G.; Klintworth, H.M.; Kavanagh, T.J.; Costa, L.G.

    2008-01-01

    In mouse cerebellar granule neurons (CGN) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (−/−) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase, phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs. PMID:18164102

  5. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    PubMed Central

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  6. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels.

    PubMed

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry; Lee, C Justin

    2016-04-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  7. Wounding Induces the Rapid and Transient Activation of a Specific MAP Kinase Pathway.

    PubMed Central

    Bogre, L.; Ligterink, W.; Meskiene, I.; Barker, P. J.; Heberle-Bors, E.; Huskisson, N. S.; Hirt, H.

    1997-01-01

    Mechanical injury in plants induces responses that are involved not only in healing but also in defense against a potential pathogen. To understand the intracellular signaling mechanism of wounding, we have investigated the involvement of protein kinases. Using specific antibodies, we showed that wounding alfalfa leaves specifically induces the transient activation of the p44MMK4 kinase, which belongs to the family of mitogen-activated protein kinases. Whereas activation of the MMK4 pathway is a post-translational process and was not blocked by [alpha]-amanitin and cycloheximide, inactivation depends on de novo transcription and translation of a protein factor(s). After wound-induced activation, the MMK4 pathway was subject to a refractory period of 25 min, during which time restimulation was not possible, indicating that the inactivation mechanism is only transiently active. After activation of the p44MMK4 kinase by wounding, transcript levels of the MMK4 gene increased, suggesting that the MMK4 gene may be a direct target of the MMK4 pathway. In contrast, transcripts of the wound-inducible MsWIP gene, encoding a putative proteinase inhibitor, were detected only several hours after wounding. Abscisic acid, methyl jasmonic acid, and electrical activity are known to mediate wound signaling in plants. However, none of these factors was able to activate the p44MMK4 kinase in the absence of wounding, suggesting that the MMK4 pathway acts independently of these signals. PMID:12237344

  8. MAP kinase dynamics in yeast.

    PubMed

    van Drogen, F; Peter, M

    2001-09-01

    MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP-kinase signaling. PMID:11730324

  9. MAP kinase mediates epidermal growth factor- and phorbol ester-induced prostacyclin formation in cardiomyocytes.

    PubMed

    Braconi Quintaje, S; Rebsamen, M; Church, D J; Vallotton, M B; Lang, U

    1998-05-01

    We studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in epidermal growth factor (EGF)-induced prostacyclin (PGI2) production in cultured, spontaneously-beating neonatal ventricular rat cardiomyocytes. To this purpose, the effect of EGF on cardiomyocyte MAPK phosphorylation, MAPK activity and PGI2-production were investigated, and compared to those induced by the PKC activator 4 beta phorbol 12-myristate 13-acetate (PMA). Both EGF (0.1 microM) and PMA (0.1 microM) induced the rapid and reversible phosphorylation of 42 KDa-MAPK in ventricular cardiomyocytes, responses that were accompanied by transient increases in MAPK activity (190-230% of control values within 5 min), and two- to three-fold increases in PGI2 formation. The tyrosine kinase inhibitors lavendustin (1 microM) and genistein (10 microM) strongly inhibited EGF-induced MAPK activation and PGI2-formation, but had no effect on PMA-stimulated responses. Experiments with the PKC inhibitor CGP 41251 (1 microM) or with PKC-downregulated cells demonstrated that in contrast to the PMA-stimulated responses, EGF-induced MAPK activation and PGI2-production were PKC-independent processes. Investigating the role of MAPK in EGF- and in PMA-promoted PGI2-formation, we found that the MAPK-inhibitor 6-thioguanine (500 microM), as well as the MAPK-kinase-inhibitor PD98059 (50 microM) abolished both EGF- and PMA-stimulated PGI2-production in cardiomyocytes. Our results indicate that MAPK-activation is at the basis of both growth factor receptor and PKC-dependent eicosanoid-formation in ventricular cardiomyocytes, where EGF-induced prostaglandin-production takes place via a PKC-independent pathway. PMID:9618234

  10. Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

    PubMed Central

    Nebreda, A R; Gannon, J V; Hunt, T

    1995-01-01

    The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone. Images PMID:8521817

  11. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

    PubMed Central

    Grimsey, Neil J.; Aguilar, Berenice; Smith, Thomas H.; Le, Phillip; Soohoo, Amanda L.; Puthenveedu, Manojkumar A.; Nizet, Victor

    2015-01-01

    Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption. PMID:26391660

  12. Intermittent Hypoxia-Induced Spinal Inflammation Impairs Respiratory Motor Plasticity by a Spinal p38 MAP Kinase-Dependent Mechanism

    PubMed Central

    Huxtable, Adrianne G.; Smith, Stephanie M.C.; Peterson, Timothy J.; Watters, Jyoti J.

    2015-01-01

    Inflammation is characteristic of most clinical disorders that challenge the neural control of breathing. Since inflammation modulates neuroplasticity, we studied the impact of inflammation caused by prolonged intermittent hypoxia on an important form of respiratory plasticity, acute intermittent hypoxia (three, 5 min hypoxic episodes, 5 min normoxic intervals) induced phrenic long-term facilitation (pLTF). Because chronic intermittent hypoxia elicits neuroinflammation and pLTF is undermined by lipopolysaccharide-induced systemic inflammation, we hypothesized that one night of intermittent hypoxia (IH-1) elicits spinal inflammation, thereby impairing pLTF by a p38 MAP kinase-dependent mechanism. pLTF and spinal inflammation were assessed in anesthetized rats pretreated with IH-1 (2 min hypoxia, 2 min normoxia; 8 h) or sham normoxia and allowed 16 h for recovery. IH-1 (1) transiently increased IL-6 (1.5 ± 0.2-fold; p = 0.02) and inducible nitric oxide synthase (iNOS) (2.4 ± 0.4-fold; p = 0.01) mRNA in cervical spinal homogenates, (2) elicited a sustained increase in IL-1β mRNA (2.4 ± 0.2-fold; p < 0.001) in isolated cervical spinal microglia, and (3) abolished pLTF (−1 ± 5% vs 56 ± 10% in controls; p < 0.001). pLTF was restored after IH-1 by systemic NSAID administration (ketoprofen; 55 ± 9%; p < 0.001) or spinal p38 MAP kinase inhibition (58 ± 2%; p < 0.001). IH-1 increased phosphorylated (activated) p38 MAP kinase immunofluorescence in identified phrenic motoneurons and adjacent microglia. In conclusion, IH-1 elicits spinal inflammation and impairs pLTF by a spinal p38 MAP kinase-dependent mechanism. By targeting inflammation, we may develop strategies to manipulate respiratory motor plasticity for therapeutic advantage when the respiratory control system is compromised (e.g., sleep apnea, apnea of prematurity, spinal injury, or motor neuron disease). PMID:25926462

  13. Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

    PubMed

    Lopachev, Alexander V; Lopacheva, Olga M; Osipova, Ekaterina A; Vladychenskaya, Elizaveta A; Smolyaninova, Larisa V; Fedorova, Tatiana N; Koroleva, Olga V; Akkuratov, Evgeny E

    2016-07-01

    Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27338714

  14. Enterobacter sakazakii targets DC-SIGN to induce immunosuppressive responses in dendritic cells by modulating MAP kinases

    PubMed Central

    Mittal, Rahul; Bulgheresi, Silvia; Emami, Claudia; Prasadarao, Nemani V.

    2009-01-01

    Enterobacter sakazakii (ES) is an emerging pathogen that causes meningitis and necrotizing enterocolitis in infants. Dendritic cells (DCs) are professional phagocytic cells that play an essential role in host defense against invading pathogens, however, the interaction of ES with DCs is not known. Here, we demonstrate that ES targets DC-SIGN to survive in myeloid DCs for which outer membrane protein A (OmpA) expression in ES is critical, although it is not required for uptake. In addition, DC-SIGN expression was sufficient to cause a significant invasion by ES in HeLa cells and intestinal epithelial cells, which are normally not invaded by ES. OmpA+ ES prevented the maturation of DCs by triggering the production of high levels of IL-10 and TGF-β and by suppressing the activation of MAP kinases. Pretreatment of DCs with antibodies to IL-10 and TGF-β or of bacteria with anti-OmpA antibodies significantly enhanced the maturation markers on DCs. Furthermore, DCs pretreated with various inhibitors of MAP kinases prohibited the increased production of pro-inflammatory cytokines stimulated by LPS or OmpA− ES. LPS pretreatment followed by OmpA+ ES infection of DCs failed to induce maturation of DCs, indicating that OmpA+ ES renders the cells in immunosuppressive state to external stimuli. Similarly, OmpA+ ES infected DCs failed to present antigen to T cells as indicated by the inability of T cells to proliferate in mixed lymphocyte reaction. We conclude that ES interacts with DC-SIGN to subvert the host immune responses by disarming MAP kinase pathway in DCs. PMID:19846880

  15. Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

    PubMed Central

    Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

  16. Role played by Disabled-2 in albumin induced MAP Kinase signalling

    SciTech Connect

    Diwakar, Ramaswamy Pearson, Alexander L.; Colville-Nash, Paul; Baines, Deborah L.; Dockrell, Mark E.C.

    2008-02-15

    Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGF{beta}-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundance in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGF{beta}-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGF{beta}-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.

  17. Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling.

    PubMed

    Tsioumpekou, Maria; Papadopoulos, Natalia; Burovic, Fatima; Heldin, Carl-Henrik; Lennartsson, Johan

    2016-09-01

    Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase Cγ and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The co-operation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKCζ interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling. PMID:27339033

  18. Functional aspects of the EGF-induced MAP kinase cascade: a complex self-organizing system approach.

    PubMed

    Kosmidis, Efstratios K; Moschou, Vasiliki; Ziogas, Georgios; Boukovinas, Ioannis; Albani, Maria; Laskaris, Nikolaos A

    2014-01-01

    The EGF-induced MAP kinase cascade is one of the most important and best characterized networks in intracellular signalling. It has a vital role in the development and maturation of living organisms. However, when deregulated, it is involved in the onset of a number of diseases. Based on a computational model describing a "surface" and an "internalized" parallel route, we use systems biology techniques to characterize aspects of the network's functional organization. We examine the re-organization of protein groups from low to high external stimulation, define functional groups of proteins within the network, determine the parameter best encoding for input intensity and predict the effect of protein removal to the system's output response. Extensive functional re-organization of proteins is observed in the lower end of stimulus concentrations. As we move to higher concentrations the variability is less pronounced. 6 functional groups have emerged from a consensus clustering approach, reflecting different dynamical aspects of the network. Mutual information investigation revealed that the maximum activation rate of the two output proteins best encodes for stimulus intensity. Removal of each protein of the network resulted in a range of graded effects, from complete silencing to intense activation. Our results provide a new "vista" of the EGF-induced MAP kinase cascade, from the perspective of complex self-organizing systems. Functional grouping of the proteins reveals an organizational scheme contrasting the current understanding of modular topology. The six identified groups may provide the means to experimentally follow the dynamics of this complex network. Also, the vulnerability analysis approach may be used for the development of novel therapeutic targets in the context of personalized medicine. PMID:25372488

  19. Functional Aspects of the EGF-Induced MAP Kinase Cascade: A Complex Self-Organizing System Approach

    PubMed Central

    Kosmidis, Efstratios K.; Moschou, Vasiliki; Ziogas, Georgios; Boukovinas, Ioannis; Albani, Maria; Laskaris, Nikolaos A.

    2014-01-01

    The EGF-induced MAP kinase cascade is one of the most important and best characterized networks in intracellular signalling. It has a vital role in the development and maturation of living organisms. However, when deregulated, it is involved in the onset of a number of diseases. Based on a computational model describing a “surface” and an “internalized” parallel route, we use systems biology techniques to characterize aspects of the network’s functional organization. We examine the re-organization of protein groups from low to high external stimulation, define functional groups of proteins within the network, determine the parameter best encoding for input intensity and predict the effect of protein removal to the system’s output response. Extensive functional re-organization of proteins is observed in the lower end of stimulus concentrations. As we move to higher concentrations the variability is less pronounced. 6 functional groups have emerged from a consensus clustering approach, reflecting different dynamical aspects of the network. Mutual information investigation revealed that the maximum activation rate of the two output proteins best encodes for stimulus intensity. Removal of each protein of the network resulted in a range of graded effects, from complete silencing to intense activation. Our results provide a new “vista” of the EGF-induced MAP kinase cascade, from the perspective of complex self-organizing systems. Functional grouping of the proteins reveals an organizational scheme contrasting the current understanding of modular topology. The six identified groups may provide the means to experimentally follow the dynamics of this complex network. Also, the vulnerability analysis approach may be used for the development of novel therapeutic targets in the context of personalized medicine. PMID:25372488

  20. MAP kinase cascades: scaffolding signal specificity.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-01-22

    Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways. PMID:11818078

  1. A Mathematical Exploration of MAP Kinase Behavior

    NASA Astrophysics Data System (ADS)

    Adams, Rhys; Balazsi, Gabor

    2008-03-01

    Mitogen-Activated Protein (MAP) kinase pathways are highly conserved from yeast to humans and are implicated in cell survival and cell death. Signaling through these pathways starts with the phosphorylation of the most upstream component (MAP kinase kinase kinase, MAPKKK), continues with phosphorylation of a MAP kinase kinase (MAPKK), and ends with phosphorylation of the target MAP kinase (MAPK). Theoretical studies over the past few decades have generated important insights into the dynamical behavior and signal processing capability of these pathways, including bistability, oscillations, signal amplification, etc. Prompted by the possibility of complex behavior in simpler signaling units than a full MAP kinase pathway, we investigate the possibility of In-Band Detection (IBD) within a single step of the cascade. We show that a basal rate of target phosphorylation can lead to IBD in a simpler system than the one described before, and define a precise relationship between the various reaction rates that is necessary to obtain IBD.

  2. TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

    PubMed

    Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

    2016-09-15

    Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. PMID:26993379

  3. Rutin inhibits UVB radiation-induced expression of COX-2 and iNOS in hairless mouse skin: p38 MAP kinase and JNK as potential targets.

    PubMed

    Choi, Ki-Seok; Kundu, Joydeb Kumar; Chun, Kyung-Soo; Na, Hye-Kyung; Surh, Young-Joon

    2014-10-01

    Exposure to ultraviolet B (UVB) radiation, a complete environmental carcinogen, induces oxidative and inflammatory skin damage, thereby increasing the risk of skin carcinogenesis. The antioxidant and anti-inflammatory activities of a wide variety of plant polyphenols have been reported. Rutin (3-rhamnosyl-glucosylquercetin), a polyphenol present in many edible plants, possesses diverse pharmacological properties including antioxidant, anti-inflammatory, antimutagenic and anticancer activities. The present study was aimed to investigate the effects of rutin on UVB-induced inflammation in mouse skin in vivo. Topical application of rutin onto the dorsal skin of female HR-1 hairless mice 30 min prior to UVB irradiation diminished epidermal hyperplasia and the levels of proteins modified by 4-hydroxynonenal, which is a biochemical hallmark of lipid peroxidation. Topical application of rutin also significantly inhibited UVB-induced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), two representative inflammatory enzymes, in hairless mouse skin. Rutin inhibited the DNA binding of activator protein-1 (AP-1) and phosphorylation of signal transducer and activator of transcription-3 (STAT3) in mouse skin exposed to UVB. Moreover, rutin attenuated UVB-induced phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun-N-terminal kinase (JNK). Pharmacological inhibition of p38 MAP kinase and JNK decreased UVB-induced expression of COX-2 in mouse skin. Taken together, these findings suggest that rutin exerts anti-inflammatory effects in UVB-irradiated mouse skin by inhibiting expression of COX-2 and iNOS, which is attributable to its suppression of p38 MAP kinase and JNK signaling responsible for AP-1 activation. PMID:24875145

  4. Cerium oxide nanoparticles inhibit lipopolysaccharide induced MAP kinase/NF-kB mediated severe sepsis.

    PubMed

    Selvaraj, Vellaisamy; Nepal, Niraj; Rogers, Steven; Manne, Nandini D P K; Arvapalli, Ravikumar; Rice, Kevin M; Asano, Shinichi; Fankenhanel, Erin; Ma, J Y; Shokuhfar, Tolou; Maheshwari, Mani; Blough, Eric R

    2015-09-01

    The life threatening disease of sepsis is associated with high mortality. Septic patient survivability with currently available treatments has failed to improve. The purpose of this study was to evaluate whether lipopolysaccharide (LPS) induced sepsis mortality and associated hepatic dysfunction can be prevented by cerium oxide nanoparticles (CeO2NPs) treatment in male Sprague Dawley rats. Here we provide the information about the methods processing of raw data related to our study published in Biomaterials (Selvaraj et al., Biomaterials, 2015, In press) and Data in Brief (Selvaraj et al., Data in Brief, 2015, In Press). The data present here provides confirmation of cerium oxide nanoparticle treatments ability to prevent the LPS induced sepsis associated changes in physiological, blood cell count, inflammatory protein and growth factors in vivo. In vitro assays investigation the treated of macrophages cells with different concentrations of cerium oxide nanoparticle demonstrate that concentration of cerium oxide nanoparticles below 1 µg/ml did not significantly influence cell survival as determined by the MTT assay. PMID:26217772

  5. A superoxide anion-scavenger, 1,3-selenazolidin-4-one suppresses serum deprivation-induced apoptosis in PC12 cells by activating MAP kinase

    SciTech Connect

    Nishina, Atsuyoshi; Kimura, Hirokazu; Kozawa, Kunihisa; Sommen, Geoffroy; Nakamura, Takao; Heimgartner, Heinz; Koketsu, Mamoru; Furukawa, Shoei

    2011-12-15

    Synthetic organic selenium compounds, such as ebselen, may show glutathione peroxidase-like antioxidant activity and have a neurotrophic effect. We synthesized 1,3-selenazolidin-4-ones, new types of synthetic organic selenium compounds (five-member ring compounds), to study their possible applications as antioxidants or neurotrophic-like molecules. Their superoxide radical scavenging effects were assessed using the quantitative, highly sensitive method of real-time kinetic chemiluminescence. At 166 {mu}M, the O{sub 2}{sup -} scavenging activity of 1,3-selenazolidin-4-ones ranged from 0 to 66.2%. 2-[3-(4-Methoxyphenyl)-4-oxo-1,3-selenazolidin-2-ylidene]malononitrile (compound b) showed the strongest superoxide anion-scavenging activity among the 6 kinds of 2-methylene-1,3-selenazolidin-4-ones examined. Compound b had a 50% inhibitory concentration (IC{sub 50}) at 92.4 {mu}M and acted as an effective and potentially useful O{sub 2}{sup -} scavenger in vitro. The effect of compound b on rat pheochromocytome cell line PC12 cells was compared with that of ebselen or nerve growth factor (NGF) by use of the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. When ebselen was added at 100 {mu}M or more, toxicity toward PC12 cells was evident. On the contrary, compound b suppressed serum deprivation-induced apoptosis in PC12 cells more effectively at a concentration of 100 {mu}M. The activity of compound b to phosphorylate mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) 1/2 (MAP kinase) in PC12 cells was higher than that of ebselen, and the former at 100 {mu}M induced the phosphorylation of MAP kinase to a degree similar to that induced by NGF. From these results, we conclude that this superoxide anion-scavenger, compound b, suppressed serum deprivation-induced apoptosis by promoting the phosphorylation of MAP kinase. -- Highlights: Black-Right-Pointing-Pointer We newly synthesized 1,3-selenazolidin-4-ones to

  6. The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFκB signalling pathways in monocytes/macrophages

    PubMed Central

    Hatziieremia, S; Gray, A I; Ferro, V A; Paul, A; Plevin, R

    2006-01-01

    Background and purpose: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. Experimental approaches: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFκB pathway, at the level of IκBα degradation and phosphorylation of NFκB. Also its effects on NFκB and GAS/GAF-DNA binding were assessed by EMSA. Key results: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IκBα or phosphorylation of NFκB. However, it inhibited NFκB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNγ-stimulated iNOS induction and GAS/GAF-DNA binding. Conclusions and Implications: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFκB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified. PMID:16894344

  7. Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

    SciTech Connect

    Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja; Joseph, Binoy; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Yin, Yuanqin; Roy, Ram Vinod; Lu, Jian; Zhang, Zhuo; Wang, Yitao; and others

    2014-10-01

    Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE{sub 2} and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE{sub 2} production. • C3G

  8. A novel PPAR{gamma} agonist, KR62776, suppresses RANKL-induced osteoclast differentiation and activity by inhibiting MAP kinase pathways

    SciTech Connect

    Park, Ju-Young; Bae, Myung-Ae; Cheon, Hyae Gyeong; Kim, Sung Soo; Hong, Jung-Min; Kim, Tae-Ho; Choi, Je-Yong; Kim, Sang-Hyun; Lim, Jiwon; Choi, Chang-Hyuk; Shin, Hong-In; Kim, Shin-Yoon Park, Eui Kyun

    2009-01-16

    We investigated the effects of a novel peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, KR62776, on osteoclast differentiation and function, and on the underlying signaling pathways. KR62776 markedly suppressed differentiation into osteoclasts in various osteoclast model systems, including bone marrow mononuclear (BMM) cells and a co-culture of calvarial osteoblasts and BMM cells. KR62776 suppressed the activation of tartrate-resistant acid phosphatase (TRAP) and the expression of genes associated with osteoclast differentiation, such as TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated receptor (OSCAR). Furthermore, KR62776 reduced resorption pit formation in osteoclasts, and down-regulated genes essential for osteoclast activity, such as Src and {alpha}v{beta}3 integrin. An analysis of a signaling pathway showed that KR62776 inhibited the receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and nuclear factor-{kappa}B (NF-{kappa}B). Together, these results demonstrate that KR62776 negatively affects osteoclast differentiation and activity by inhibiting the RANKL-induced activation of MAP kinases and NF-{kappa}B.

  9. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signalling pathways in SKH-1 mice skin

    PubMed Central

    Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

    2015-01-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm2) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicates that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. PMID:25680589

  10. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin.

    PubMed

    Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

    2015-04-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm(2)) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. PMID:25680589

  11. β-Adrenergic stimulation does not activate p38 MAP kinase or induce PGC-1α in skeletal muscle.

    PubMed

    Kim, Sang Hyun; Asaka, Meiko; Higashida, Kazuhiko; Takahashi, Yumiko; Holloszy, John O; Han, Dong-Ho

    2013-04-15

    There are reports that the β-adrenergic agonist clenbuterol induces a large increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle. This has led to the hypothesis that the increases in PGC-1α and mitochondrial biogenesis induced in muscle by endurance exercise are mediated by catecholamines. In the present study, we evaluated this possibility and found that injecting rats with clenbuterol or norepinephrine induced large increases in PGC-1α and mitochondrial proteins in brown adipose tissue but had no effect on PGC-1α expression or mitochondrial biogenesis in skeletal muscle. In brown adipocytes, the increase in PGC-1α expression induced by β-adrenergic stimulation is mediated by activation of p38 mitogen-activated protein kinase (p38 MAPK), which phosphorylates and activates the cAMP response element binding protein (CREB) family member activating transcription factor 2 (ATF2), which binds to a cyclic AMP response element (CRE) in the PGC-1α promoter and mediates the increase in PGC-1α transcription. Phospho-CREB does not have this effect. Our results show that the reason for the lack of effect of β-adrenergic stimulation on PGC-1α expression in muscle is that catecholamines do not activate p38 or increase ATF2 phosphorylation in muscle. PMID:23443926

  12. Aloe-Emodin Induces Chondrogenic Differentiation of ATDC5 Cells via MAP Kinases and BMP-2 Signaling Pathways.

    PubMed

    Yang, Ming; Li, Liang; Heo, Seok-Mo; Soh, Yunjo

    2016-07-01

    Endochondral bone formation is the process by which mesenchymal cells condense into chondrocytes, which are ultimately responsible for new bone formation. The processes of chondrogenic differentiation and hypertrophy are critical for bone formation and are therefore highly regulated. The present study was designed to investigate the effect of aloe-emodin on chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Aloe-emodin treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. ATDC5 cells were treated with aloe-emodin and stained with alcian blue. Compared with the control cells, the ATDC5 cells showed more intense alcian blue staining. This finding suggested that aloe-emodin induced the synthesis of matrix proteoglycans and increased the activity of alkaline phosphatase. Aloe-emodin also enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, BSP and RunX2 in a time-dependent manner. Furthermore, examination of the MAPK signaling pathway showed that aloe-emodin increased the activation of extracellular signal-regulated kinase (ERK), but had no effect on p38 and c-jun N-terminal kinase (JNK). Aloe-emodin also enhanced the protein expression of BMP-2 in a time-dependent manner. Thus, these results showed that aloe-emodin exhibited chodromodulating effects via the BMP-2 or ERK signaling pathway. Aloe-emodin may have potential future applications for the treatment of growth disorders. PMID:27350340

  13. Aloe-Emodin Induces Chondrogenic Differentiation of ATDC5 Cells via MAP Kinases and BMP-2 Signaling Pathways

    PubMed Central

    Yang, Ming; Li, Liang; Heo, Seok-Mo; Soh, Yunjo

    2016-01-01

    Endochondral bone formation is the process by which mesenchymal cells condense into chondrocytes, which are ultimately responsible for new bone formation. The processes of chondrogenic differentiation and hypertrophy are critical for bone formation and are therefore highly regulated. The present study was designed to investigate the effect of aloe-emodin on chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Aloe-emodin treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. ATDC5 cells were treated with aloe-emodin and stained with alcian blue. Compared with the control cells, the ATDC5 cells showed more intense alcian blue staining. This finding suggested that aloe-emodin induced the synthesis of matrix proteoglycans and increased the activity of alkaline phosphatase. Aloe-emodin also enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, BSP and RunX2 in a time-dependent manner. Furthermore, examination of the MAPK signaling pathway showed that aloe-emodin increased the activation of extracellular signal-regulated kinase (ERK), but had no effect on p38 and c-jun N-terminal kinase (JNK). Aloe-emodin also enhanced the protein expression of BMP-2 in a time-dependent manner. Thus, these results showed that aloe-emodin exhibited chodromodulating effects via the BMP-2 or ERK signaling pathway. Aloe-emodin may have potential future applications for the treatment of growth disorders. PMID:27350340

  14. Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase.

    PubMed

    Mithoe, Sharon C; Ludwig, Christina; Pel, Michiel J C; Cucinotta, Mara; Casartelli, Alberto; Mbengue, Malick; Sklenar, Jan; Derbyshire, Paul; Robatzek, Silke; Pieterse, Corné M J; Aebersold, Ruedi; Menke, Frank L H

    2016-03-01

    Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex. PMID:26769563

  15. MAP4K Family Kinases in Immunity and Inflammation.

    PubMed

    Chuang, Huai-Chia; Wang, Xiaohong; Tan, Tse-Hua

    2016-01-01

    MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases. MAP4Ks including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK have been reported to induce JNK activation through activating the MAP3K-MAP2K cascade. The physiological roles of MAP4Ks in immunity and inflammation are largely unknown until recent studies using biochemical approaches and knockout mice. Surprisingly, JNK is not the major target of MAP4Ks in immune cells; MAP4Ks regulate immune responses through novel targets. HPK1 inhibits T-cell receptor (TCR) signaling and B-cell receptor signaling via inducing phosphorylation/ubiquitination of SLP-76 and BLNK, respectively. GLK activates TCR signaling through phosphorylating/activating PKCθ. T-cell-mediated immune responses and Th17-mediated experimental autoimmune diseases are enhanced in HPK1 knockout mice but ameliorated in GLK knockout mice. Consistently, HPK1 levels are decreased in peripheral blood mononuclear cells and T cells from patients with psoriatic arthritis and systemic lupus erythematosus (SLE), respectively. Moreover, GLK levels are increased in T cells from patients with SLE, rheumatoid arthritis, or adult-onset Still's disease; the percentages of GLK-overexpression T cells are correlated with the disease activity. In addition, HGK phosphorylates and induces TRAF2 protein degradation, leading to negative regulation of IL-6 production in resting T cells. Loss of HGK in T cells results in spontaneous systemic inflammation and type 2 diabetes in mice. HGK is also involved in cancer cell migration. To date, the phenotypes of knockout mice for GCK, KHS, and MINK have not been reported; the roles of these three MAP4Ks in immune cell signaling are discussed in this review. Taken together, MAP4K family kinases play diverse roles in immune cell signaling, immune responses, and inflammation. PMID:26791862

  16. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin

    SciTech Connect

    Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Hitron, John Andrew; Wang, Lei; Asha, Padmaja; Shi, Xianglin; Zhang, Zhuo

    2015-04-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm{sup 2}) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E{sub 2} (PGE{sub 2}), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. - Highlights: • Blackberry extract inhibits UVB-induced glutathione depletion.

  17. Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

    PubMed

    Romanov, Victor; Whyard, Terry C; Waltzer, Wayne C; Grollman, Arthur P; Rosenquist, Thomas

    2015-01-01

    Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells. PMID:24792323

  18. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    PubMed

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

    cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation. PMID:26335579

  19. 22(R)-hydroxycholesterol induces HuR-dependent MAP kinase phosphatase-1 expression via mGluR5-mediated Ca(2+)/PKCα signaling.

    PubMed

    Kim, Hyunmi; Woo, Joo Hong; Lee, Jee Hoon; Joe, Eun-Hye; Jou, Ilo

    2016-08-01

    MAP kinase phosphatase (MKP)-1 plays a pivotal role in controlling MAP kinase (MAPK)-dependent (patho) physiological processes. Although MKP-1 gene expression is tightly regulated at multiple levels, the underlying mechanistic details remain largely unknown. In this study, we demonstrate that MKP-1 expression is regulated at the post-transcriptional level by 22(R)-hydroxycholesterol [22(R)-HC] through a novel mechanism. 22(R)-HC induces Hu antigen R (HuR) phosphorylation, cytoplasmic translocation and binding to MKP-1 mRNA, resulting in stabilization of MKP-1 mRNA. The resulting increase in MKP-1 leads to suppression of JNK-mediated inflammatory responses in brain astrocytes. We further demonstrate that 22(R)-HC-induced phosphorylation of nuclear HuR is mediated by PKCα, which is activated in the cytosol by increases in intracellular Ca(2+) levels mediated by the phospholipase C/inositol 1,4,5-triphosphate receptor (PLC/IP3R) pathway and translocates from cytoplasm to nucleus. In addition, pharmacological interventions reveal that metabotropic glutamate receptor5 (mGluR5) is responsible for the increases in intracellular Ca(2+) that underlie these actions of 22(R)-HC. Collectively, our findings identify a novel anti-inflammatory mechanism of 22(R)-HC, which acts through PKCα-mediated cytoplasmic shuttling of HuR to post-transcriptionally regulate MKP-1 expression. These findings provide an experimental basis for the development of a RNA-targeted therapeutic agent to control MAPK-dependent inflammatory responses. PMID:27206966

  20. Involvement of the H1 histamine receptor, p38 MAP kinase, MLCK, and Rho/ROCK in histamine-induced endothelial barrier dysfunction

    PubMed Central

    Adderley, Shaquria P.; Zhang, Xun E.; Breslin, Jerome W.

    2015-01-01

    Objective The mechanisms by which histamine increases microvascular permeability remain poorly understood. We tested the hypothesis that H1 receptor activation disrupts the endothelial barrier and investigated potential downstream signals. Methods We used confluent endothelial cell (EC) monolayers, assessing transendothelial electrical resistance (TER) as an index of barrier function. Human umbilical vein EC (HUVEC), cardiac microvascular EC (HCMEC), and dermal microvascular EC (HDMEC) were compared. Receptor expression was investigated using Western blotting, immunofluorescence (IF) confocal microscopy and RT-PCR. Receptor function and downstream signaling pathways were tested using pharmacologic antagonists and inhibitors, respectively. Results We identified H1-H4 receptors on all three EC types. H1 antagonists did not affect basal TER but prevented the histamine-induced decrease in TER. Blockade of H2 or H3 attenuated the histamine response only in HDMEC, while inhibition of H4 attenuated the response only in HUVEC. Combined inhibition of both PKC and PI3K caused exaggerated histamine-induced barrier dysfunction in HDMEC, whereas inhibition of p38 MAP kinase attenuated the histamine response in all three EC types. Inhibition of RhoA, ROCK, or MLCK also prevented the histamine-induced decrease in TER in HDMEC. Conclusion The data suggest that multiple signaling pathways contribute to histamine-induced endothelial barrier dysfunction via the H1 receptor. PMID:25582918

  1. Salicylic acid activates a 48-kD MAP kinase in tobacco.

    PubMed Central

    Zhang, S; Klessig, D F

    1997-01-01

    The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses. PMID:9165755

  2. FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2.

    PubMed Central

    Tan, Y; Rouse, J; Zhang, A; Cariati, S; Cohen, P; Comb, M J

    1996-01-01

    Fibroblast growth factor (FGF) activates a protein kinase cascade in SK-N-MC cells that regulates gene expression at a cyclic-AMP response element (CRE) by stimulating the transcriptional activity of CREB. The activation of CREB is prevented by a dominant negative mutant of Ras and triggered via the same site (Ser133) that becomes phosphorylated in response to cyclic AMP and Ca2+. However, the effect of FGF is not mediated by cyclic AMP-dependent protein kinase, TPA-sensitive isoforms of protein kinase-C, p70S6K or p90rsk (all of which phosphorylate CREB at Ser133 in vitro). Instead, we identify the FGF-stimulated CREB kinase as MAP kinase-activated protein (MAPKAP) kinase-2, an enzyme that lies immediately downstream of p38 MAP kinase, in a pathway that is also stimulated by cellular stresses. We show that MAPKAP kinase-2 phosphorylates CREB at Ser133 in vitro, that the FGF- or stress-induced activation of MAPKAP kinase-2 and phosphorylation of CREB and ATF-1 are prevented by similar concentrations of the specific p38 MAP kinase inhibitor SB 203580, and that MAPKAP kinase-2 is the only detectable SB 203580-sensitive CREB kinase in SK-N-MC cell extracts. We also show that transfection of RK/p38 MAP kinase in SK-N-MC cells, but not transfection of p44 MAP kinase, activates Gal4-CREB-dependent transcription via Ser133. These findings identify a new growth factor and stress-activated signaling pathway that regulates gene expression at the CRE. Images PMID:8887554

  3. A novel antitubulin agent, DPQZ, induces cell apoptosis in human oral cancer cells through Ras/Raf inhibition and MAP kinases activation.

    PubMed

    Hour, Mann-Jen; Lee, Kun-Tsung; Wu, Yang-Chang; Wu, Chi-Yu; You, Bang-Jau; Chen, Tai-Lin; Lee, Hong-Zin

    2013-05-01

    6-(N,N-Dimethylamino)-2-(naphthalene-1-yl)-4-quinazolinone (DPQZ)-induced apoptosis was accompanied by the characteristics of DNA fragmentation and phosphatidylserine externalization in human oral cancer HSC-3 cells. The IC50 (half maximal inhibitory concentration) value of DPQZ is about 0.25 μM at 24 h. The interference in the dynamics of tubulin and cell division of DPQZ, like vinblastine (0.01 μM), has been proven in this study. Treatment of HSC-3 cells with DPQZ resulted in many of mitotic cells with multipolar spindles. Up-regulation of MAP kinases, such as ERK, JNK, and p38, mediated by DPQZ appears to be involved in DPQZ-induced apoptosis in HSC-3 cells. It is worthy of note that the expression of Ras and c-Raf that lie upstream of ERK were inhibited by DPQZ. In addition, the DPQZ-induced cell death was attenuated by JNK inhibitor SP600125 (3 or 10 μM), not by the ERK or p38 inhibitors. JNK inhibitor abolished the DPQZ-induced increase in the phosphorylation of Bcl-2 and the protein levels of proform caspase-3, caspase-8, and caspase-9, indicating that JNK is an upstream activator of Bcl-2 and caspase family members and plays a key role in DPQZ-induced HSC-3 cell apoptosis. We also attempted to develop an anticancer drug that is designed to kill rapidly dividing cancer cells while causing less damage to normal cells. The DPQZ-induced cytotoxicity against human gingival fibroblasts was less than that against HSC-3 cells. Our work provides a new strategy and mechanism for developing anticancer drug and may contribute to clinical anticancer drug discovery and application. PMID:23212307

  4. Isoflavonoid-Rich Flemingia macrophylla Extract Attenuates UVB-Induced Skin Damage by Scavenging Reactive Oxygen Species and Inhibiting MAP Kinase and MMP Expression

    PubMed Central

    Chiang, Hsiu-Mei; Chiu, Hua-Hsien; Liao, Sue-Tsai; Chen, Yen-Ting; Chang, Hsien-Chang; Wen, Kuo-Ching

    2013-01-01

    In this study, we investigated the antioxidant activity and anti-photoaging properties of an extract of Flemingia macrophylla, a plant rich in isoflavonoid content. Pretreatment of fibroblasts with Flemingia macrophylla extract (FME) inhibited elastase activity, promoted the protein expression of type I procollagen, and attenuated the phosphorylation of mitogen-activated protein (MAP) kinase and the protein expression of matrix-metalloproteinase- (MMP-) 1, 3, and 9. The IC50 values were 2.1 μg/mL for DPPH radical scavenging ability, 366.8 μg/mL for superoxide anion scavenging ability, 178.9 μg/mL for hydrogen peroxide scavenging ability, and 230.9 μg/mL for hydroxyl radical scavenging ability. Also, exposure of erythrocytes to various concentrations of FME (50–500 μg/mL) resulted in a dose- and time-dependent inhibition of AAPH-induced hemolysis. In human fibroblasts, FME at 10 μg/mL was shown to be a potent scavenger of UV-induced reactive oxygen species (ROS). The antioxidant and anti-photoaging properties of FME make it an ideal anti-intrinsic aging and anti-photoaging agent. PMID:23935672

  5. Isoflavonoid-Rich Flemingia macrophylla Extract Attenuates UVB-Induced Skin Damage by Scavenging Reactive Oxygen Species and Inhibiting MAP Kinase and MMP Expression.

    PubMed

    Chiang, Hsiu-Mei; Chiu, Hua-Hsien; Liao, Sue-Tsai; Chen, Yen-Ting; Chang, Hsien-Chang; Wen, Kuo-Ching

    2013-01-01

    In this study, we investigated the antioxidant activity and anti-photoaging properties of an extract of Flemingia macrophylla, a plant rich in isoflavonoid content. Pretreatment of fibroblasts with Flemingia macrophylla extract (FME) inhibited elastase activity, promoted the protein expression of type I procollagen, and attenuated the phosphorylation of mitogen-activated protein (MAP) kinase and the protein expression of matrix-metalloproteinase- (MMP-) 1, 3, and 9. The IC50 values were 2.1  μ g/mL for DPPH radical scavenging ability, 366.8  μ g/mL for superoxide anion scavenging ability, 178.9  μ g/mL for hydrogen peroxide scavenging ability, and 230.9  μ g/mL for hydroxyl radical scavenging ability. Also, exposure of erythrocytes to various concentrations of FME (50-500  μ g/mL) resulted in a dose- and time-dependent inhibition of AAPH-induced hemolysis. In human fibroblasts, FME at 10  μ g/mL was shown to be a potent scavenger of UV-induced reactive oxygen species (ROS). The antioxidant and anti-photoaging properties of FME make it an ideal anti-intrinsic aging and anti-photoaging agent. PMID:23935672

  6. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms.

    PubMed

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  7. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms

    PubMed Central

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  8. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) integrates developmental signals for eyelid closure

    PubMed Central

    Geh, Esmond; Meng, Qinghang; Mongan, Maureen; Wang, Jingcai; Takatori, Atsushi; Zheng, Yi; Puga, Alvaro; Lang, Richard A.; Xia, Ying

    2011-01-01

    Developmental eyelid closure is an evolutionarily conserved morphogenetic event requiring proliferation, differentiation, cytoskeleton reorganization, and migration of epithelial cells at the tip of the developing eyelid. Many signaling events take place during eyelid closure, but how the signals converge to regulate the morphogenetic process remains an open and intriguing question. Here we show that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) highly expressed in the developing eyelid epithelium, forms with c-Jun, a regulatory axis that orchestrates morphogenesis by integrating two different networks of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one of these networks by inducing c-Jun expression which, in a phosphorylation-independent manner, binds to the Map3k1 promoter and causes an increase in MAP3K1 expression. RhoA knockout in the ocular surface epithelium disturbs this network by decreasing MAP3K1 expression, and causes delayed eyelid closure in Map3k1 hemizygotes. The second network is initiated by the enzymatic activity of MAP3K1, which phosphorylates and activates a JNK-c-Jun module, leading to AP-1 transactivation and induction of its downstream genes, such as Pai-1. MAP3K1 inactivation reduces AP-1 activity and PAI-1 expression both in cells and developing eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 pathways in developmental eyelid closure. PMID:21969564

  9. S. aureus haemolysin A-induced IL-8 and IL-6 release from human airway epithelial cells is mediated by activation of p38- and Erk-MAP kinases and additional, cell type-specific signalling mechanisms.

    PubMed

    Räth, Susann; Ziesemer, Sabine; Witte, Amelie; Konkel, Anne; Müller, Christian; Hildebrandt, Petra; Völker, Uwe; Hildebrandt, Jan-Peter

    2013-07-01

    Soluble virulence-associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o-, S9 or A549 cells with recombinant Hla (rHla). In a dose-dependent manner, rHla induced secretion of IL-8 in all three cell types, but IL-6 release only in 16HBE14o- and S9 cells. rHla-mediated secretion of IL-8 and IL-6 was suppressed by pre-incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL-8 release in all three cell types and for IL-6 release in 16HBE14o- and S9 cells. The rHla-mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca(2+)]i, an early response in rHla-treated cells. Inhibitors of calmodulin or calcium/calmodulin-dependent kinase II attenuated rHla-mediated release of IL-8 in 16HBE14o- and A549 cells and of IL-6 in 16HBE14o- cells. This indicates that rHla may mediate simultaneous activation of calmodulin-dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin-dependent signalling did not affect rHla-induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase. PMID:23347173

  10. Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling.

    PubMed

    Kim, Junwon; Rose, Mark D

    2015-12-01

    Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth

  11. Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling

    PubMed Central

    Kim, Junwon; Rose, Mark D.

    2015-01-01

    Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth

  12. Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative, on Osteoclast Differentiation, Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway

    PubMed Central

    Ihn, Hye Jung; Lee, Doohyun; Lee, Taeho; Kim, Sang-Hyun; Shin, Hong-In; Bae, Yong Chul; Hong, Jung Min; Park, Eui Kyun

    2015-01-01

    Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss. PMID:26536233

  13. Reversal of the TPA-induced inhibition of gap junctional intercellular communication by Chaga mushroom (Inonotus obliquus) extracts: effects on MAP kinases.

    PubMed

    Park, Jung-Ran; Park, Joon-Suk; Jo, Eun-Hye; Hwang, Jae-Woong; Kim, Sun-Jung; Ra, Jeong-Chan; Aruoma, Okezie I; Lee, Yong-Soon; Kang, Kyung-Sun

    2006-01-01

    Chaga mushroom (Inonotus obliquus) has continued to receive attention as a folk medicine with indications for the treatment of cancers and digestive diseases. The anticarcinogenic effect of Chaga mushroom extract was investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 normal rat liver epithelial cells. The cells were pre-incubated with Chaga mushroom extracts (5, 10, 20 microg/ml) for 24 h and this was followed by co-treatment with Chaga mushroom extracts and TPA (12-O-tetradecanoylphorbol-13-acetate, 10 ng/ml) for 1 h. The inhibition of GJIC by TPA (12-O-tetradecanoylphorbol-13-acetate), promoter of cancer, was prevented with treatment of Chaga mushroom extracts. Similarly, the increased phosphorylated ERK1/2 and p38 protein kinases were markedly reduced in Chaga mushroom extracts-treated cells. There was no change in the JNK kinase protein level, suggesting that Chaga mushroom extracts could only block the activation of ERK1/2 and p38 MAP kinase. The Chaga mushroom extracts further prevented the inhibition of GJIC through the blocking of Cx43 phosphorylation. Indeed cell-to-cell communication through gap junctional channels is a critical factor in the life and death balance of cells because GJIC has an important function in maintaining tissue homeostasis through the regulation of cell growth, differentiation, apoptosis and adaptive functions of differentiated cells. Thus Chaga mushroom may act as a natural anticancer product by preventing the inhibition of GJIC through the inactivation of ERK1/2 and p38 MAP kinase. PMID:17012771

  14. FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

    PubMed Central

    Worthen, G S; Avdi, N; Buhl, A M; Suzuki, N; Johnson, G L

    1994-01-01

    Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants. Images PMID:8040337

  15. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

    SciTech Connect

    Chou, C.-T.; He Shiping; Jan, C.-R. . E-mail: crjan@isca.vghks.gov.tw

    2007-02-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.

  16. Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

    PubMed Central

    Xing, Bin; Xin, Tao; Hunter, Randy Lee; Bing, Guoying

    2008-01-01

    Background Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone. Methods Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures. Results Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on

  17. MAP kinase-interacting kinases--emerging targets against cancer.

    PubMed

    Diab, Sarah; Kumarasiri, Malika; Yu, Mingfeng; Teo, Theodosia; Proud, Christopher; Milne, Robert; Wang, Shudong

    2014-04-24

    Mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) regulate the initiation of translation through phosphorylation of eukaryotic initiation factor 4E (eIF4E). Mnk-mediated eIF4E activation promotes cancer development and progression. While the phosphorylation of eIF4E is necessary for oncogenic transformation, the kinase activity of Mnks seems dispensable for normal development. For this reason, pharmacological inhibition of Mnks could represent an ideal mechanism-based and nontoxic therapeutic strategy for cancer treatment. In this review, we discuss the current understanding of Mnk biological roles, structures, and functions, as well as clinical implications. Importantly, we propose different strategies for identification of highly selective small molecule inhibitors of Mnks, including exploring a structural feature of their kinase domain, DFD motif, which is unique within the human kinome. We also argue that a combined targeting of Mnks and other pathways should be considered given the complexity of cancer. PMID:24613018

  18. Protective effects of green tea polyphenol extracts against ethanol-induced gastric mucosal damages in rats: stress-responsive transcription factors and MAP kinases as potential targets.

    PubMed

    Lee, Jeong-Sang; Oh, Tae-Young; Kim, Young-Kyung; Baik, Joo-Hyun; So, Sung; Hahm, Ki-Baik; Surh, Young-Joon

    2005-11-11

    There are multiple lines of compelling evidence from epidemiologic and laboratory studies supporting that frequent consumption of green tea is inversely associated with the risk of chronic human diseases including cancer. The chemopreventive and chemoprotective effects of green tea have been largely attributed to antioxidative and anti-inflammatory activities of its polyphenolic constituents, such as epigallocatechin gallate. The present study was designed to evaluate the efficacy of green tea polyphenols in protecting against alcohol-induced gastric damage and to elucidate the underlying mechanisms. Intragastric administration of ethanol to male Sprague-Dawley rats caused significant gastric mucosal damage, which was accompanied by elevated expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as transient activation of redox-sensitive transcription factors, such as NF-kappaB and AP-1, and mitogen-activated protein kinases (MAPKs). Oral administration of the green tea polyphenolic extract (GTE) significantly ameliorated mucosal damages induced by ethanol and also attenuated the ethanol-induced expression of COX-2 and iNOS. Inactivation of MAPKs, especially p38 and ERKl/2, by GTE might be responsible for inhibition of ethanol-induced expression of COX-2 and iNOS. PMID:16095631

  19. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  20. Mangiferin, a Natural Xanthone, Protects Murine Liver in Pb(II) Induced Hepatic Damage and Cell Death via MAP Kinase, NF-κB and Mitochondria Dependent Pathways

    PubMed Central

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2013-01-01

    One of the most well-known naturally occurring environmental heavy metals, lead (Pb) has been reported to cause liver injury and cellular apoptosis by disturbing the prooxidant-antioxidant balance via oxidative stress. Several studies, on the other hand, reported that mangiferin, a naturally occurring xanthone, has been used for a broad range of therapeutic purposes. In the present study, we, therefore, investigated the molecular mechanisms of the protective action of mangiferin against lead-induced hepatic pathophysiology. Lead [Pb(II)] in the form of Pb(NO3)2 (at a dose of 5 mg/kg body weight, 6 days, orally) induced oxidative stress, hepatic dysfunction and cell death in murine liver. Post treatment of mangiferin at a dose of 100 mg/kg body weight (6 days, orally), on the other hand, diminished the formation of reactive oxygen species (ROS) and reduced the levels of serum marker enzymes [alanine aminotranferase (ALT) and alkaline phosphatase (ALP)]. Mangiferin also reduced Pb(II) induced alterations in antioxidant machineries, restored the mitochondrial membrane potential as well as mutual regulation of Bcl-2/Bax. Furthermore, mangiferin inhibited Pb(II)-induced activation of mitogen-activated protein kinases (MAPKs) (phospho-ERK 1/2, phosphor-JNK phospho- p38), nuclear translocation of NF-κB and apoptotic cell death as was evidenced by DNA fragmentation, FACS analysis and histological assessment. In vitro studies using hepatocytes as the working model also showed the protective effect of mangiferin in Pb(II) induced cytotoxicity. All these beneficial effects of mangiferin contributes to the considerable reduction of apoptotic hepatic cell death induced by Pb(II). Overall results demonstrate that mangiferin exhibit both antioxidative and antiapoptotic properties and protects the organ in Pb(II) induced hepatic dysfunction. PMID:23451106

  1. 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

  2. Abscisic Acid-Induced Resistance against the Brown Spot Pathogen Cochliobolus miyabeanus in Rice Involves MAP Kinase-Mediated Repression of Ethylene Signaling1[C][W][OA

    PubMed Central

    De Vleesschauwer, David; Yang, Yinong; Vera Cruz, Casiana; Höfte, Monica

    2010-01-01

    The plant hormone abscisic acid (ABA) is involved in an array of plant processes, including the regulation of gene expression during adaptive responses to various environmental cues. Apart from its well-established role in abiotic stress adaptation, emerging evidence indicates that ABA is also prominently involved in the regulation and integration of pathogen defense responses. Here, we demonstrate that exogenously administered ABA enhances basal resistance of rice (Oryza sativa) against the brown spot-causing ascomycete Cochliobolus miyabeanus. Microscopic analysis of early infection events in control and ABA-treated plants revealed that this ABA-inducible resistance (ABA-IR) is based on restriction of fungal progression in the mesophyll. We also show that ABA-IR does not rely on boosted expression of salicylic acid-, jasmonic acid -, or callose-dependent resistance mechanisms but, instead, requires a functional Gα-protein. In addition, several lines of evidence are presented suggesting that ABA steers its positive effect on brown spot resistance through antagonistic cross talk with the ethylene (ET) response pathway. Exogenous ethephon application enhances susceptibility, whereas genetic disruption of ET signaling renders plants less vulnerable to C. miyabeanus attack, thereby inducing a level of resistance similar to that observed on ABA-treated wild-type plants. Moreover, ABA treatment alleviates C. miyabeanus-induced activation of the ET reporter gene EBP89, while derepression of pathogen-triggered EBP89 transcription via RNA interference-mediated knockdown of OsMPK5, an ABA-primed mitogen-activated protein kinase gene, compromises ABA-IR. Collectively, these data favor a model whereby exogenous ABA enhances resistance against C. miyabeanus at least in part by suppressing pathogen-induced ET action in an OsMPK5-dependent manner. PMID:20130100

  3. Human p38{delta} MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

    SciTech Connect

    Ozawa, Shigeyuki; Ito, Shin; Kato, Yasumasa; Kubota, Eiro; Hata, Ryu-Ichiro

    2010-06-11

    The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38{alpha}, {beta}, {gamma} and {delta}. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38{alpha} and {beta}, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38{gamma} and/or {delta} was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38{delta} attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38{delta} with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38{delta} isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38{alpha} and/or {beta} isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

  4. Deguelin-induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down-regulation of fibroblast growth factor receptor 4 activity.

    PubMed

    Wu, Wei; Hai, Yang; Chen, Lu; Liu, Rui-Jin; Han, Yu-Xiang; Li, Wen-Hao; Li, Song; Lin, Shuo; Wu, Xin-Rong

    2016-04-01

    Deguelin, a natural component derived from leguminous plants, has been used as pesticide in some regions. Accumulating evidence show that deguelin has promising chemopreventive and therapeutic activities against cancer cells. This study shows that low concentrations of deguelin can lead to significant delay in zebrafish embryonic development through growth inhibition and induction of apoptosis. Furthermore, we identified fibroblast growth factor receptor 4 (FGFR4) as the putative target of deguelin. The candidate was initially identified by a microarray approach and then validated through in vitro experiments using hormone-responsive (MCF-7) and nonresponsive (MDA-MB-231) human breast cancer cell lines. The results show that deguelin suppressed cell proliferation and induced apoptosis in both cancer cell lines, but not in Hs 578Bst cells, by blocking PI3K/AKT and mitogen-activated protein kinases (MAPK) signaling. The FGFR4 mRNA and protein level also diminished in a dose-dependent manner. Interestingly, we found that forced FGFR4 overexpression attenuated deguelin-induced proliferative suppression and apoptotic cell death in both zebrafish and MCF-7 cell lines, p-AKT and p-ERK levels were restored upon FGFR4 overexpression. Taken together, our results strongly suggest that deguelin inhibition of PI3K/AKT and MAPK signaling in zebrafish and breast cancer cell lines is partially mediated through down-regulation of FGFR4 activity. PMID:27069628

  5. The role of mitogen-activated protein kinase in oxytocin-induced contraction of uterine smooth muscle in pregnant rat.

    PubMed

    Nohara, A; Ohmichi, M; Koike, K; Masumoto, N; Kobayashi, M; Akahane, M; Ikegami, H; Hirota, K; Miyake, A; Murata, Y

    1996-12-24

    Oxytocin causes the rapid tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in both human and rat puerperal uterine myometrial cultured cells. The potential role of the MAP kinase pathway in oxytocin action was investigated with the specific MAP kinase kinase (MEK) inhibitor, PD98059. Oxytocin stimulation of the tyrosine phosphorylation of MAP kinase in both human and rat cultured puerperal uterine cells was abolished by pretreatment of the cells with MEK inhibitor in a dose-dependent manner. Although MEK inhibitor had no effect on oxytocin-induced intracellular Ca2+ mobilization in either pregnant human or pregnant rat uterine cells, it partly inhibited oxytocin-induced pregnant rat uterine contraction in a dose-dependent manner. These results suggest that MAP kinase pathway may have some important roles in oxytocin-induced uterine contraction. PMID:8954997

  6. Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle.

    PubMed

    Larsen, J K; Yamboliev, I A; Weber, L A; Gerthoffer, W T

    1997-11-01

    The 27-kDa heat shock protein (HSP27) is expressed in a variety of tissues in the absence of stress and is thought to regulate actin filament dynamics, possibly by a phosphorylation/dephosphorylation mechanism. HSP27 has also been suggested to be involved in contraction of intestinal smooth muscle. We have investigated phosphorylation of HSP27 in airway smooth muscle in response to the muscarinic agonist carbachol. Carbachol increased 32P incorporation into canine tracheal HSP27 and induced a shift in the distribution of charge isoforms on two-dimensional gels to more acidic, phosphorylated forms. The canine HSP27 amino acid sequence includes three serine residues corresponding to sites in human HSP27 known to be phosphorylated by mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2. To determine whether muscarinic receptors are coupled to a "stress response" pathway in smooth muscle culminating in phosphorylation of HSP27, we assayed MAPKAP kinase-2 activity and tyrosine phosphorylation of p38 mitogen-activated protein (MAP) kinase, the enzyme thought to activate MAPKAP kinase-2. Recombinant canine HSP27 expressed in Escherichia coli was a substrate for MAPKAP kinase-2 in vitro as well as a substrate for endogenous smooth muscle HSP27 kinase, which was activated by carbachol. Carbachol also increased tyrosine phosphorylation of p38 MAP kinase. SB-203580, an inhibitor of p38 MAP kinases, reduced activation of endogenous HSP27 kinase activity and blocked the shift in HSP27 charge isoforms to acidic forms. We suggest that HSP27 in airway smooth muscle, in addition to being a stress response protein, is phosphorylated by a receptor-initiated signaling cascade involving muscarinic receptors, tyrosine phosphorylation of p38 MAP kinase, and activation of MAPKAP kinase-2. PMID:9374719

  7. Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4

    PubMed Central

    Varin, E M; Wojtusciszyn, A; Broca, C; Muller, D; Ravier, M A; Ceppo, F; Renard, E; Tanti, J-F; Dalle, S

    2016-01-01

    Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells. PMID:26794660

  8. Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

    PubMed Central

    Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

    2015-01-01

    Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

  9. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

    PubMed

    Millar, J B; Buck, V; Wilkinson, M G

    1995-09-01

    Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast. PMID:7657164

  10. Redundancy in the World of MAP Kinases: All for One

    PubMed Central

    Saba-El-Leil, Marc K.; Frémin, Christophe; Meloche, Sylvain

    2016-01-01

    The protein kinases ERK1 and ERK2 are the effector components of the prototypical ERK1/2 mitogen-activated protein (MAP) kinase pathway. This signaling pathway regulates cell proliferation, differentiation and survival, and is essential for embryonic development and cellular homeostasis. ERK1 and ERK2 homologs share similar biochemical properties but whether they exert specific physiological functions or act redundantly has been a matter of controversy. However, recent studies now provide compelling evidence in support of functionally redundant roles of ERK1 and ERK2 in embryonic development and physiology. In this review, we present a critical assessment of the evidence for the functional specificity or redundancy of MAP kinase isoforms. We focus on the ERK1/ERK2 pathway but also discuss the case of JNK and p38 isoforms. PMID:27446918

  11. Acetaldehyde alters MAP kinase signalling and epigenetic histone modifications in hepatocytes.

    PubMed

    Shukla, Shivendra D; Lee, Youn Ju; Park, Pil-hoon; Aroor, Annayya R

    2007-01-01

    Although both oxidative and non-oxidative metabolites of ethanol are involved in generating ethanol matabolic stress (Emess), the oxidative metabolite acetaldehyde plays a critical role in the cellular actions of ethanol. We have investigated the effects of acetaldehyde on p42/44 MAP kinase, p46/p54 c-jun N-terminal kinase (JNK1/JNK2) and p38 MAP kinase in hepatocytes. Acetaldehyde caused temporal activation of p42/44 MAPK followed by JNK, but the activation of the p42/44 MAPK was not a prerequisite for the JNK activation. Activation ofJNK1 by acetaldehyde was greater than JNK2. Ethanol and acetaldehyde activatedJNK have opposing roles; ethanol-induced JNK activation increased apoptosis whereas that by acetaldehyde decreased apoptosis. Acetaldehyde also caused histone H3 acetylation at Lys9 and phosphorylation of histone H3 at Serl0 and 28, the latter being dependent on p38 MAP kinase. Phosphorylation at Ser28 was higher than at Serl0. Thus acetaldehyde distinctively alters MAP kinase signalling and histone modifications, processes involved in transcriptional activation. PMID:17590997

  12. Prolonged activation of mitogen-activated protein kinases during NSAID-induced apoptosis in HT-29 colon cancer cells.

    PubMed

    Kim, T I; Jin, S H; Kim, W H; Kang, E H; Choi, K Y; Kim, H J; Shin, S K; Kang, J K

    2001-06-01

    The mechanisms of the antineoplastic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) still are unknown, but the induction of apoptosis is one of the possible mechanisms. We attempted to demonstrate the role of mitogen-activated protein (MAP) kinases, generally considered to be important mediators of proliferative and apoptotic signals, in NSAID-induced colon cancer cell apoptosis. Apoptosis was detected by demonstration of DNA fragmentation in agarose gel electrophoresis. Cell death was assessed by trypan blue dye exclusion method. MAP kinase activation was assessed by Western blot using phosphospecific antibodies to MAP kinases. Kinase assay using activating transcription factor-2 (ATF-2) fusion protein as a substrate was also performed for measuring p38 MAP kinase activity. For the inhibition of p38 MAP kinase, pyridinylimidazole compound (SB203580) was utilized. Caspase-3 activity was measured using the tetrapeptide fluorogenic substrate Ac-DEVD-AMC. Treatment of HT-29 cells with NSAIDs results in time- and dose-dependent induction of apoptosis, accompanied by sustained activation of all three MAP kinase subfamilies. The SB203580, a p38 MAP kinase inhibitor, reduced indomethacin-induced cell death by 43%, while PD098059, a MAPK/ERK kinase (MEK)1 inhibitor, did not affect cell death. p38 MAP kinase and caspase-3 activation were not significantly interlinked in indomethacin-induced apoptosis. From these results, we conclude that NSAIDs can induce prolonged activation of MAP kinases in colon cancer cells and that, of these, p38 MAP kinase may play a partial but significant role in indomethacin-induced apoptosis. PMID:11459290

  13. Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

    PubMed Central

    Jeffree, Chris E.; Oborny, Radek; Boonyarungsrit, Patid; Read, Nick D.

    2012-01-01

    In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia. PMID:22900028

  14. Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

    SciTech Connect

    Xing, Li; Shieh, Huey S.; Selness, Shaun R.; Devraj, Rajesh V.; Walker, John K.; Devadas, Balekudru; Hope, Heidi R.; Compton, Robert P.; Schindler, John F.; Hirsch, Jeffrey L.; Benson, Alan G.; Kurumbail, Ravi G.; Stegeman, Roderick A.; Williams, Jennifer M.; Broadus, Richard M.; Walden, Zara; Monahan, Joseph B.; Pfizer

    2009-07-24

    PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.

  15. Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

    PubMed

    Châtel, A; Hamer, B; Talarmin, H; Dorange, G; Schröder, H C; Müller, W E G

    2010-03-01

    Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution. PMID:19948362

  16. Mechanical Impact Induces Cartilage Degradation via Mitogen Activated Protein Kinases

    PubMed Central

    Ding, Lei; Heying, Emily; Nicholson, Nathan; Stroud, Nicolas J.; Homandberg, Gene A.; Guo, Danping; Buckwalter, Joseph A.; Martin, James A.

    2010-01-01

    Objective To determine the activation of MAP kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction induced chondrocyte death and cartilage degeneration. Design The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan loss were determined with fluorescent microscopy and DMMB assay. The expression of catabolic genes at mRNA levels was examined with quantitative real time PCR. Results Early p38 activation was detected at 20 min and 1 hr post-impaction. At 24 hr, enhanced phosphorylation of p38 and ERK1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and proteoglycan losses. Quantative Real-time PCR analysis revealed that blunt impaction significantly up-regulated MMP-13, TNF-α, and ADAMTS-5 expression. Conclusion These findings implicate p38 and ERK MAPKs in the post injury spread of cartilage degeneration and suggest that the risk of PTOA following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α. PMID:20813194

  17. Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways.

    PubMed

    Abdulnour, Raja-Elie E; Peng, Xinqi; Finigan, Jay H; Han, Eugenia J; Hasan, Emile J; Birukov, Konstantin G; Reddy, Sekhar P; Watkins, James E; Kayyali, Usamah S; Garcia, Joe G N; Tuder, Rubin M; Hassoun, Paul M

    2006-09-01

    Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI. PMID:16632522

  18. Rapamycin enhances eIF4E phosphorylation by activating MAP kinase-interacting kinase 2a (Mnk2a).

    PubMed

    Stead, Rebecca L; Proud, Christopher G

    2013-08-19

    Eukaryotic initiation factor eIF4E and its phosphorylation play key roles in cell transformation and tumorigenesis. eIF4E is phosphorylated by the Mnks (MAP (mitogen-activated protein) kinase-interacting kinases). Rapamycin increases eIF4E phosphorylation in cancer cells, potentially limiting their anti-cancer effects. Here we show that the rapamycin-induced increase in eIF4E phosphorylation reflects increased activity of Mnk2 but not Mnk1. This activation requires a novel phosphorylation site in Mnk2a, Ser437. Our findings have potentially important implications for the use of rapamycin and its analogues in cancer therapy, suggesting that inhibitors of mTOR and Mnk (or Mnk2) may be more efficacious than rapalogs alone. PMID:23831578

  19. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  20. MAP Kinase Pathways: Molecular Roads to Primary Acral Lentiginous Melanoma.

    PubMed

    Fernandes, Juliana D; Hsieh, Ricardo; de Freitas, Luiz A R; Brandao, Miguel A R; Lourenço, Silvia V; Sangueza, Martin; Nico, Marcello M S

    2015-12-01

    The etiology and pathogenesis of lentiginous acral melanomas are poorly understood. Recent studies have postulated that DNA repair mechanisms and cell growth pathways are involved in the development of melanoma, particularly changes in the MAPK pathways (RAS, BRAF, MEK 1/2, and ERK 1/2). The aim of this study is to assess the status of the MAP kinase pathways in the pathogenesis of acral melanomas. The authors examined the components of the RAS-RAF-MEK-ERK cascades by immunohistochemistry in a series of 16 primary acral melanomas by tissue microarray. The expression of MAP kinase cascade proteins changed in most cases. The authors observed that 57.14% of cases were BRAF positive and that 61.53%, 71.42%, and 71.42% of cases were positive for MEK2, ERK1, and ERK2, respectively; RAS was not expressed in 92.31%, and all cases were negative for MEK1. The absence of RAS and positivity for MEK2, ERK1, and ERK2 were most seen in invasive cases with high thickness. These aspects of the MAPK pathway require further examination in acral melanomas between different populations. Nevertheless, the results highlight significant alterations in the MAP kinase cascades that are related to histological indicators of prognosis in primary acral melanomas. PMID:26588333

  1. MAP Kinase Pathways: Molecular Roads to Primary Acral Lentiginous Melanoma

    PubMed Central

    Hsieh, Ricardo; de Freitas, Luiz A. R.; Brandao, Miguel A. R.; Lourenço, Silvia V.; Sangueza, Martin; Nico, Marcello M. S.

    2015-01-01

    Abstract: The etiology and pathogenesis of lentiginous acral melanomas are poorly understood. Recent studies have postulated that DNA repair mechanisms and cell growth pathways are involved in the development of melanoma, particularly changes in the MAPK pathways (RAS, BRAF, MEK 1/2, and ERK 1/2). The aim of this study is to assess the status of the MAP kinase pathways in the pathogenesis of acral melanomas. The authors examined the components of the RAS–RAF–MEK–ERK cascades by immunohistochemistry in a series of 16 primary acral melanomas by tissue microarray. The expression of MAP kinase cascade proteins changed in most cases. The authors observed that 57.14% of cases were BRAF positive and that 61.53%, 71.42%, and 71.42% of cases were positive for MEK2, ERK1, and ERK2, respectively; RAS was not expressed in 92.31%, and all cases were negative for MEK1. The absence of RAS and positivity for MEK2, ERK1, and ERK2 were most seen in invasive cases with high thickness. These aspects of the MAPK pathway require further examination in acral melanomas between different populations. Nevertheless, the results highlight significant alterations in the MAP kinase cascades that are related to histological indicators of prognosis in primary acral melanomas. PMID:26588333

  2. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  3. Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.

    PubMed Central

    Hehl, S; Stoyanov, B; Oehrl, W; Schönherr, R; Wetzker, R; Heinemann, S H

    2001-01-01

    Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect. PMID:11736661

  4. L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons

    PubMed Central

    Kolaj, Miloslav; Zhang, Li

    2016-01-01

    In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca2+) contribution to the components of this TRH-induced response. TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca2+]o, suggesting a critical role for both intracellular Ca2+ release and Ca2+ influx. TRH-induced inward current was unchanged by T-type Ca2+ channel blockade, but was decreased by blockade of high-voltage-activated Ca2+ channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca2+ influx via L-type Ca2+ channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca2+-induced Ca2+ release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca2+ influx via opening of L-type Ca2+ channels, a sensitivity of a TRPC-like component to intracellular Ca2+ release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component. PMID:27009047

  5. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  6. BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival

    PubMed Central

    Jacque, Emilie; Schweighoffer, Edina; Tybulewicz, Victor L.J.

    2015-01-01

    B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase–Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers. PMID:25987726

  7. Inhibition of MAP kinase/NF-kB mediated signaling and attenuation of lipopolysaccharide induced severe sepsis by cerium oxide nanoparticles

    PubMed Central

    Selvaraj, Vellaisamy; Nepal, Niraj; Rogers, Steven; Manne, Nandini D.P.K.; Arvapalli, Ravikumar; Rice, Kevin M.; Asano, Shinichi; Fankhanel, Erin; Ma, Jane J.; Shokuhfar, Tolou; Maheshwari, Mani; Blough, Eric R.

    2015-01-01

    Sepsis is a life threatening disease that is associated with high mortality. Existing treatments have failed to improve survivability in septic patients. The purpose of this present study is to evaluate whether cerium oxide nanoparticles (CeO2NPs) can prevent lipopolysaccharide (LPS) induced severe sepsis mortality by preventing hepatic dysfunction in male Sprague Dawley rats. Administration of a single dose (0.5 mg/kg) of CeO2NPs intravenously to septic rats significantly improved survival rates and functioned to restore body temperature, respiratory rate and blood pressure towards baseline. Treatment-induced increases in animal survivability were associated with decreased hepatic damage along with reductions in serum cytokines/chemokines, and diminished inflammatory related signaling. Kupffer cells and macrophage cells exposed to CeO2NPs exhibited decreases in LPS-induced cytokine release (TNF-α, IL-1β, IL-6, HMGB1) which were associated with diminished cellular ROS, reduced levels of nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and decreased nuclear factor-kappa light chain enhancer of activated B cells (NF-kB) transcriptional activity. The findings of this study indicate that CeO2NPs may be useful as a therapeutic agent for sepsis. PMID:25968464

  8. Inhibition of MAP kinase/NF-kB mediated signaling and attenuation of lipopolysaccharide induced severe sepsis by cerium oxide nanoparticles.

    PubMed

    Selvaraj, Vellaisamy; Nepal, Niraj; Rogers, Steven; Manne, Nandini D P K; Arvapalli, Ravikumar; Rice, Kevin M; Asano, Shinichi; Fankhanel, Erin; Ma, Jane J; Shokuhfar, Tolou; Maheshwari, Mani; Blough, Eric R

    2015-08-01

    Sepsis is a life threatening disease that is associated with high mortality. Existing treatments have failed to improve survivability in septic patients. The purpose of this present study is to evaluate whether cerium oxide nanoparticles (CeO2NPs) can prevent lipopolysaccharide (LPS) induced severe sepsis mortality by preventing hepatic dysfunction in male Sprague Dawley rats. Administration of a single dose (0.5 mg/kg) of CeO2NPs intravenously to septic rats significantly improved survival rates and functioned to restore body temperature, respiratory rate and blood pressure towards baseline. Treatment-induced increases in animal survivability were associated with decreased hepatic damage along with reductions in serum cytokines/chemokines, and diminished inflammatory related signaling. Kupffer cells and macrophage cells exposed to CeO2NPs exhibited decreases in LPS-induced cytokine release (TNF-α, IL-1β, IL-6, HMGB1) which were associated with diminished cellular ROS, reduced levels of nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and decreased nuclear factor-kappa light chain enhancer of activated B cells (NF-kB) transcriptional activity. The findings of this study indicate that CeO2NPs may be useful as a therapeutic agent for sepsis. PMID:25968464

  9. Ixora parviflora Protects against UVB-Induced Photoaging by Inhibiting the Expression of MMPs, MAP Kinases, and COX-2 and by Promoting Type I Procollagen Synthesis

    PubMed Central

    Wen, Kuo-Ching; Fan, Pei-Ching; Tsai, Shang-Yuan; Shih, I-Chen; Chiang, Hsiu-Mei

    2012-01-01

    Ixora parviflora with high polyphenol content exhibited antioxidant activity and reducing UVB-induced intracellular reactive oxygen species production. In this study, results of the photoaging screening experiments revealed that IPE at 1000 μg/mL reduced the activity of bacterial collagenase by 92.7 ± 4.2% and reduced the activity of elastase by 32.6 ± 1.4%. Therefore, we investigated the mechanisms by which IPE exerts its anti-photoaging activity. IPE at 1 μg/mL led to an increase in type I procollagen expression and increased total collagen synthesis in fibroblasts at 5 μg/mL. We found that IPE inhibited MMP-1, MMP-3, and MMP-9 expression at doses of 1, 5, and 10 μg/mL, respectively, in fibroblasts exposed to UV irradiation (40 mJ/cm2). Gelatin zymography assay showed that IPE at 50 μg/mL inhibited MMP-9 secretion/activity in cultured fibroblasts after UVB exposure. In addition, IPE inhibited the phosphorylation of p38, ERK, and JNK induced by UVB. Furthermore, IPE inhibited the UVB-induced expression of Smad7. In addition, IPE at 1 μg/mL inhibited NO production and COX-2 expression in UV-exposed fibroblasts. These findings show that IPE exhibits anti-inflammatory and anti-photoaging activities, indicating that IPE could be a potential anti-aging agent. PMID:22203872

  10. Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

    PubMed Central

    Hellesøy, Monica; Lorens, James B.

    2015-01-01

    The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

  11. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  12. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  13. A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase.

    PubMed

    Torada, Atsushi; Koike, Michiya; Ogawa, Taiichi; Takenouchi, Yu; Tadamura, Kazuki; Wu, Jianzhong; Matsumoto, Takashi; Kawaura, Kanako; Ogihara, Yasunari

    2016-03-21

    Seed germination under the appropriate environmental conditions is important both for plant species survival and for successful agriculture. Seed dormancy, which controls germination time, is one of the adaptation mechanisms and domestication traits [1]. Seed dormancy is generally defined as the absence of germination of a viable seed under conditions that are favorable for germination [2]. The seed dormancy of cultivated plants has generally been reduced during domestication [3]. Bread wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Weak dormancy may be an advantage for the productivity due to uniform emergence and a disadvantage for the risks of pre-harvest sprouting (PHS), which decreases grain quality and yield [4]. A number of quantitative trait loci (QTLs) controlling natural variation of seed dormancy have been identified on various chromosomes [5]. A major QTL for seed dormancy has been consistently detected on chromosome 4A [6-13]. The QTL was designated as a major gene, Phs1, which could be precisely mapped within a 2.6 cM region [14]. Here, we identified a mitogen-activated protein kinase kinase 3 (MKK3) gene (designated TaMKK3-A) by a map-based approach as a candidate gene for the seed dormancy locus Phs1 on chromosome 4A in bread wheat. Complementation analysis showed that transformation of a dormant wheat cultivar with the TaMKK3-A allele from a nondormant cultivar clearly reduced seed dormancy. Cultivars differing in dormancy had a single nonsynonymous amino acid substitution in the kinase domain of the predicted MKK3 protein sequence, which may be associated with the length of seed dormancy. PMID:26948878

  14. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    PubMed

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons. PMID:25609615

  15. Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

    PubMed Central

    Maeda, Yukihide; Fukushima, Kunihiro; Omichi, Ryotaro; Kariya, Shin; Nishizaki, Kazunori

    2013-01-01

    Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute

  16. Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

    PubMed Central

    Groom, L A; Sneddon, A A; Alessi, D R; Dowd, S; Keyse, S M

    1996-01-01

    The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location. Images PMID:8670865

  17. The involvement of MAP kinases JNK and p38 in photodynamic injury of crayfish neurons and glial cells

    NASA Astrophysics Data System (ADS)

    Petin, Y. O.; Bibov, M. Y.; Uzdensky, A. B.

    2007-05-01

    The role of JNK and p38 MAP kinases in functional inactivation and necrosis of mechanoreceptor neurons as well as necrosis, apoptosis and proliferation of satellite glial cells induced by photodynamic treatment (10 -7 M Photosens, 30 min incubation, 670 nm laser irradiation at 0.4 W/cm2) in the isolated crayfish stretch receptor was studied using specific inhibitors SP600125 and SB202190, respectively. SP600125 enhanced PDT-induced apoptosis of photosensitized glial cells but did not influence PDT-induced changes in neuronal activity, density of glial nuclei around neuron body, and necrosis of receptor neurons and glial cells. SB202190 did not influence neuron activity and survival as well but reduced PDT-induced necrosis but not apoptosis of glial cells. Therefore, both MAP kinases influenced glial cells but not neurons. JNK protected glial cells from PDT-induced apoptosis but did not influence necrosis and proliferation of these cells. In contrast, p38 did not influence apoptosis but contributed into PDT-induced necrosis of glial cells and PDT-induced gliosis. These MAP kinase inhibitors may be used for modulation of photodynamic therapy of brain tumors.

  18. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.

    PubMed

    Lanning, Bryan R; Whitby, Landon R; Dix, Melissa M; Douhan, John; Gilbert, Adam M; Hett, Erik C; Johnson, Theodore O; Joslyn, Chris; Kath, John C; Niessen, Sherry; Roberts, Lee R; Schnute, Mark E; Wang, Chu; Hulce, Jonathan J; Wei, Baoxian; Whiteley, Laurence O; Hayward, Matthew M; Cravatt, Benjamin F

    2014-09-01

    Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors. PMID:25038787

  19. Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies.

    PubMed Central

    Yu, W.; Bozza, P. T.; Tzizik, D. M.; Gray, J. P.; Cassara, J.; Dvorak, A. M.; Weller, P. F.

    1998-01-01

    Lipid bodies are inducible lipid domains abundantly present in leukocytes engaged in inflammation. They are rich in esterified arachidonate and are also potential sites for eicosanoid-forming enzyme localization. It is therefore of interest to know whether arachidonate-releasing cytosolic phospholipase A2 (cPLA2) localizes at lipid bodies. Here, we present evidence that cPLA2 and its activating protein kinases, mitogen-activated protein (MAP) kinases, co-localize at lipid bodies. U937 cells express high levels of cPLA2 and contain numerous cytoplasmic lipid bodies. Using double-labeling immunocytochemistry we demonstrated punctate cytoplasmic localizations of both cPLA2 and MAP kinases in U937 cells that were perfectly concordant with fluorescent fatty-acid-labeled lipid bodies. The co-localization of cPLA2 and MAP kinases at lipid bodies was confirmed by subcellular fractionation and immunoblot. Lipid body fractions free of cytosol and other organelles contained significant amounts of [14C]arachidonate-labeled phosphatidylcholine and cPLA2 enzymatic activities. Immunoblotting with specific antibodies identified cPLA2 as well as MAP kinases, including ERK1, ERK2, p85, and p38, in lipid bodies. The co-compartmentalization within arachidonate-rich lipid bodies of cPLA2 and its potentially activating protein kinases suggests that lipid bodies may be structurally distinct intracellular sites active in extracellular ligand-induced arachidonate release and eicosanoid formation. Images Figure 1 Figure 2 Figure 3 Figure 6 Figure 7 PMID:9502418

  20. A mitotically inheritable unit containing a MAP kinase module

    PubMed Central

    Kicka, Sébastien; Bonnet, Crystel; Sobering, Andrew K.; Ganesan, Latha P.; Silar, Philippe

    2006-01-01

    Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate. PMID:16938837

  1. p38 MAP kinase regulates circadian rhythms in Drosophila.

    PubMed

    Vrailas-Mortimer, Alysia D; Ryan, Sarah M; Avey, Matthew J; Mortimer, Nathan T; Dowse, Harold; Sanyal, Subhabrata

    2014-12-01

    The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways. PMID:25403440

  2. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    PubMed

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  3. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways

    PubMed Central

    Yong Qiao, Xiao; Nie, Ying; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yao Yinrg, Wei; Hu, Ying; Ming Xu, Wen; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  4. Inflammation and Mechanical Stretch Promote Aortic Stiffening in Hypertension Through Activation of p38 MAP Kinase

    PubMed Central

    Wu, Jing; Thabet, Salim R.; Kirabo, Annet; Trott, Daniel W.; Saleh, Mohamed A.; Xiao, Liang; Madhur, Meena S.; Chen, Wei; Harrison, David G.

    2014-01-01

    Rationale Aortic stiffening commonly occurs in hypertension and further elevates systolic pressure. Hypertension is also associated with vascular inflammation and increased mechanical stretch. The interplay between inflammation, mechanical stretch and aortic stiffening in hypertension remains undefined. Objective To determine the role of inflammation and mechanical stretch in aortic stiffening. Methods and Results Chronic angiotensin II infusion caused marked aortic adventitial collagen deposition, as quantified by Masson’s Trichrome Blue staining and biochemically by hydroxyproline content, in wild-type (WT) but not in Recombination Activation Gene-1 deficient (RAG-1−/−) mice. Aortic compliance, defined by ex-vivo measurements of stress-strain curves, was reduced by chronic angiotensin II infusion in WT mice (p<0.01) but not in RAG-1−/− mice (p<0.05). Adoptive transfer of T cells to RAG-1−/− mice restored aortic collagen deposition and stiffness to values observed in WT mice. Mice lacking the T cell derived cytokine IL-17a were also protected against aortic stiffening. In additional studies, we found that blood pressure normalization by treatment with hydralazine and hydrochlorothiazide prevented angiotensin II-induced vascular T cell infiltration, aortic stiffening and collagen deposition. Finally, we found that mechanical stretch induces expression of collagen 1α1, 3α1 and 5a1 in cultured aortic fibroblasts in a p38 MAP kinase-dependent fashion, and that inhibition of p38 prevented angiotensin II-induced aortic stiffening in vivo. IL-17a also induced collagen 3a1 expression via activation of p38 MAP kinase. Conclusions Our data define a pathway in which inflammation and mechanical stretch lead to vascular inflammation that promotes collagen deposition. The resultant increase in aortic stiffness likely further worsens systolic hypertension and its attendant end-organ damage. PMID:24347665

  5. Dietary turmeric modulates DMBA-induced p21{sup ras}, MAP kinases and AP-1/NF-{kappa}B pathway to alter cellular responses during hamster buccal pouch carcinogenesis

    SciTech Connect

    Garg, Rachana; Ingle, Arvind; Maru, Girish

    2008-11-01

    The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-{kappa}B, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-{kappa}B DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-{kappa}B, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements.

  6. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  7. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  8. Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

    SciTech Connect

    Geiss, Brian J.; Cano, Gina L.; Tavis, John E.; Morrison, Lynda A. . E-mail: morrisla@slu.edu

    2004-12-05

    Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

  9. Polyphenol-rich pomegranate fruit extract (POMx) suppresses PMACI-induced expression of pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells

    PubMed Central

    Rasheed, Zafar; Akhtar, Nahid; Anbazhagan, Arivarasu N; Ramamurthy, Sangeetha; Shukla, Meenakshi; Haqqi, Tariq M

    2009-01-01

    Background Mast cells and basophils are multifunctional effector cells and contain plentiful secretary granules in their cytoplasm. These cell types are involved in several inflammatory and immune events and are known to produce an array of mediators including a broad spectrum of cytokines. Pomegranate fruit is rich in anthocyanins and hydrolysable tannins; a group of polyphenolic compounds shown to be potent antioxidant with anti-inflammatory activity. However, no studies have been undertaken to investigate whether a polyphenol-rich pomegranate fruit extract (POMx) inhibits the inflammatory activity of activated human mast cells and basophils. The aim of this study was to examine whether POMx modulates inflammatory reactions using human basophilic cell line KU812. Methods KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus calcium inophore A23187 (PMACI). The inhibitory effect of POMx on pro-inflammatory cytokine gene expression and production by stimulated KU812 cells was measured by quantitative RT-PCR, and cytokine-specific ELISA assays, respectively. Western blotting was used to analyze the effect of POMx on the activation of mitogen-activated protein kinases (MAPKs), and the nuclear factor (NF)-κB in PMACI stimulated KU812 cells. Effect on the activity of NF-κB was determined using Luciferase reporter assay. Significance of differences from control values were analyzed by means of standard statistical methods. Results POMx significantly decreased PMACI stimulated inflammatory gene expression and production of interleukin (IL)-6 and IL-8 in KU812 cells. The inhibitory effect of POMx on the pro-inflammatory cytokines was MAPK subgroups c-jun N-terminal kinase (JNK)- and extracellular-regulated kinase (ERK) dependent. In addition, POMx suppressed the NF-κB activation induced by PMACI by inhibiting IκB-degradation in human basophil cells. POMx also suppressed the powerful induction of NF-κB promoter-mediated luciferase activity in

  10. ANTI-APOPTOTIC ACTIONS OF VASOPRESSIN IN H32 NEURONS INVOLVE MAP KINASE TRANSACTIVATION AND BAD PHOSPHORYLATION

    PubMed Central

    Chen, Jun; Volpi, Simona; Aguilera, Greti

    2008-01-01

    Vasopressin (VP) secreted within the brain modulates neuronal function acting as a neurotransmitter. Based on the observation that VP prevented serum deprivation-induced cell death in the neuronal cell line, H32, which expresses endogenous V1 receptors, we tested the hypothesis that VP has anti-apoptotic properties. Flow cytometry experiments showed that 10nM VP prevented serum deprivation-induced cell death and annexin V binding. Serum deprivation increased caspase-3 activity in a time and serum concentration dependent manner, and VP prevented these effects through interaction with receptors of V1 subtype. The signaling pathways mediating the anti-apoptotic effect of VP involve mitogen activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK), Ca2+/calmodulin dependent kinase (CaMK) and protein kinase C (PKC). Western blot analyses revealed time-dependent decreases of Bad phosphorylation and increases in cytosolic levels of cytochrome c following serum deprivation, effects which were prevented by 10nM VP. These data demonstrate that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. The data suggest that VP has anti-apoptotic activity in neurons and that VP may act as a neuroprotective agent in the brain. PMID:18402937

  11. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  12. Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

    Park, Choon-Ho; Choi, Bo-Hwa; Jeong, Min-Woo; Kim, Sangjune; Kim, Wanil; Song, Yun Seon; Kim, Kyong-Tai

    2011-01-01

    Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. PMID:21346188

  13. Studying MAP Kinase pathways during early development of Xenopus laevis.

    PubMed

    Keren, Aviad; Bengal, Eyal

    2010-01-01

    The following chapter describes several methods involved in the detection of MAPK activities and phosphorylated proteins during early development of Xenopus laevis. The Xenopus embryo provides a powerful platform for biochemical studies. We describe here basic methods of embryo manipulations such as egg fertilization, embryo growth and maintenance, microinjection of capped RNA and antisense morpholino oligonucleotides (AMOs), and isolation of explants. In addition, we describe methods to detect phosphorylated proteins, to analyze kinase activity, and to interfere with signaling pathways. Immunohistochemical staining performed on whole embryos or on tissue sections is an additional method for the detection of phosphorylated proteins in the developing embryo. Approaches to activate or inhibit MAPK activities including the ectopic expression of mutated isoforms of MAPK kinase, or the incubation of embryo explants with pharmacological inhibitors are described. Finally, we describe an in vitro kinase assay specifically designed for the Xenopus embryo. PMID:20811998

  14. A novel lipid binding site formed by the MAP kinase insert in p38 alpha.

    PubMed

    Diskin, Ron; Engelberg, David; Livnah, Oded

    2008-01-01

    The p38 mitogen-activated protein (MAP) kinases function as signaling molecules essential for many cellular processes, particularly mediating stress response. The activity of p38 MAP kinases is meticulously regulated to reach the desired cellular phenotype. Several alternative activation and attenuation mechanisms have been characterized recently which include new phosphorylation sites. Here we present the crystal structure of p38 alpha MAP kinase in complex with n-octyl-beta-glucopyranoside detergent. The complex unveils a novel lipid-binding site formed by a local conformational change of the MAP kinase insert. This binding is the first attribution for a possible role of the MAP kinase insert in p38. The binding site can accommodate a large selection of lipidic molecules. In addition, we also show via biophysical methods that arachidonic acid and its derivatives bind p38 alpha in vitro. Based on our analysis we propose that the binding of lipids could fine-tune p38 alpha catalytic activity towards a preferred phenotype. PMID:17999933

  15. FES kinase participates in KIT-ligand induced chemotaxis

    SciTech Connect

    Voisset, Edwige; Lopez, Sophie; Chaix, Amandine; Vita, Marina; George, Coralie; Dubreuil, Patrice; De Sepulveda, Paulo

    2010-02-26

    FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.

  16. Aberrant Activation of p38 MAP Kinase-Dependent Innate Immune Responses Is Toxic to Caenorhabditis elegans

    PubMed Central

    Cheesman, Hilary K.; Feinbaum, Rhonda L.; Thekkiniath, Jose; Dowen, Robert H.; Conery, Annie L.; Pukkila-Worley, Read

    2016-01-01

    Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the nematode Caenorhabditis elegans. We previously identified an immunostimulatory xenobiotic that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the deleterious effects of the compound, and identified five toxicity suppressors. These strains contained hypomorphic mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1, and pmk-1), demonstrating that hyperstimulation of the p38 MAPK pathway is toxic to animals. To explore mechanisms of immune pathway regulation in C. elegans, we conducted another genetic screen for dominant activators of the p38 MAPK pathway, and identified a single allele that had a gain-of-function (gf) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK pmk-1. The nsy-1(gf) allele caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, had greater levels of phosphorylated p38 MAPK, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. In addition, the nsy-1(gf) mutation was toxic to developing animals. Together, these data suggest that the activity of the MAPKKK NSY-1 is tightly regulated as part of a physiological mechanism to control p38 MAPK-mediated innate immune hyperactivation, and ensure cellular homeostasis in C. elegans. PMID:26818074

  17. Aberrant Activation of p38 MAP Kinase-Dependent Innate Immune Responses Is Toxic to Caenorhabditis elegans.

    PubMed

    Cheesman, Hilary K; Feinbaum, Rhonda L; Thekkiniath, Jose; Dowen, Robert H; Conery, Annie L; Pukkila-Worley, Read

    2016-03-01

    Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the nematode Caenorhabditis elegans. We previously identified an immunostimulatory xenobiotic that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the deleterious effects of the compound, and identified five toxicity suppressors. These strains contained hypomorphic mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1, and pmk-1), demonstrating that hyperstimulation of the p38 MAPK pathway is toxic to animals. To explore mechanisms of immune pathway regulation in C. elegans, we conducted another genetic screen for dominant activators of the p38 MAPK pathway, and identified a single allele that had a gain-of-function (gf) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK pmk-1. The nsy-1(gf) allele caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, had greater levels of phosphorylated p38 MAPK, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. In addition, the nsy-1(gf) mutation was toxic to developing animals. Together, these data suggest that the activity of the MAPKKK NSY-1 is tightly regulated as part of a physiological mechanism to control p38 MAPK-mediated innate immune hyperactivation, and ensure cellular homeostasis in C. elegans. PMID:26818074

  18. The production of VEGF involving MAP kinase activation by low level laser therapy in human granulosa cells

    PubMed Central

    Utsunomiya-Kai, Yufuko; Kai, Kentaro; Miyakawa, Isao; Ohshiro, Toshio; Narahara, Hisashi

    2012-01-01

    Objective: The function of granulosa cells is regulated by various hormones and growth factors. Our aim is to clarify the regulation of vascular endothelial growth factor (VEGF) production via mitogen-activated protein kinase (MAPK) induced by low level laser therapy (LLLT) in human granulosa cells. Methods: A human granulosa cell line, KGN cells, were cultured and incubated after LLLT (60mW, GaAlAs 830nm). The levels of VEGF in the culture media were determined by an enzyme-linked immunosorbent assay. The activation of MAP kinase in KGN cells was detected by western blot analysis. Results: VEGF production was significantly increased by LLLT in a time-dependent manner. MAP kinase activity was increased by LLLT. In addition it was enhanced by LLLT and follicle-stimulating hormone (FSH) stimulation. Conclusions: The results suggested that VEGF is induced by LLLT through mechanisms involving MAPK. The increase in VEGF may contribute to neovascularization, which in turn would promote various ovulation phenomena as well as follicular growth. PMID:24511196

  19. Participation of mitogen-activated protein kinase in thapsigargin- and TPA-induced histamine production in murine macrophage RAW 264.7 cells

    PubMed Central

    Shiraishi, Muneshige; Hirasawa, Noriyasu; Kobayashi, Yuriko; Oikawa, Shinji; Murakami, Akira; Ohuchi, Kazuo

    2000-01-01

    Stimulation of the murine macrophage cell line RAW 264.7 with thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induced histamine production in a time- and concentration-dependent manner. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), also enhanced histamine production. α-Fluoromethylhistidine, a suicide substrate of L-histidine decarboxylase (HDC), suppressed the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production. Both thapsigargin (30 nM) and TPA (30 nM) induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059, a specific inhibitor of MEK-1 which phosphorylates p44/p42 MAP kinase, strongly suppressed both the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production, whereas SB203580, a specific inhibitor of p38 MAP kinase, inhibited them only partially. The other MEK-1 inhibitor, U-0126, also inhibited both the thapsigargin- and TPA-induced histamine production in a concentration-dependent manner. Thapsigargin (30 nM) and TPA (30 nM) increased the levels of HDC mRNA at 4 h, but PD98059 suppressed both the thapsigargin- and TPA-induced increases in the HDC mRNA level. These findings indicate that thapsigargin and TPA induce histamine production in RAW 264.7 cells by increasing the level of HDC mRNA, and that both the thapsigargin- and TPA-induced histamine production are regulated largely by p44/p42 MAP kinase and partially by p38 MAP kinase.. PMID:10711350

  20. Cosmic string induced CMB maps

    SciTech Connect

    Landriau, M.; Shellard, E. P. S.

    2011-02-15

    We compute maps of CMB temperature fluctuations seeded by cosmic strings using high resolution simulations of cosmic strings in a Friedmann-Robertson-Walker universe. We create full-sky, 18 deg. and 3 deg. CMB maps, including the relevant string contribution at each resolution from before recombination to today. We extract the angular power spectrum from these maps, demonstrating the importance of recombination effects. We briefly discuss the probability density function of the pixel temperatures, their skewness, and kurtosis.

  1. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth.

    PubMed

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK. PMID:19932089

  2. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    SciTech Connect

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  3. Inulin stimulates phagocytosis of PMA-treated THP-1 macrophages by involvement of PI3-kinases and MAP kinases.

    PubMed

    Nagahara, Yukitoshi; Nagamori, Taome; Tamegai, Hidekazu; Hitokuwada, Mami; Yoshimi, Yoji; Ikekita, Masahiko; Shinomiya, Takahisa

    2011-01-01

    Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation. PMID:22038771

  4. Effects of p38 MAP kinase inhibitors on the differentiation and maturation of erythroid progenitors.

    PubMed

    Dalmas, Deidre A; Tierney, Lauren A; Zhang, Cindy; Narayanan, Padma K; Boyce, Rogely W; Schwartz, Lester W; Frazier, Kendall S; Scicchitano, Marshall S

    2008-12-01

    In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1(+)cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1(+)cells after day 2 and reduced percentages of ter119(+) cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1(+)bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation. PMID:19126791

  5. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  6. MAP Kinase Pathways in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Gustin, Michael C.; Albertyn, Jacobus; Alexander, Matthew; Davenport, Kenneth

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area. PMID:9841672

  7. Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

    SciTech Connect

    Devadas, Balekudru; Selness, Shaun R.; Xing, Li; Madsen, Heather M.; Marrufo, Laura D.; Shieh, Huey; Messing, Dean M.; Yang, Jerry Z.; Morgan, Heidi M.; Anderson, Gary D.; Webb, Elizabeth G.; Zhang, Jian; Devraj, Rajesh V.; Monahan, Joseph B.

    2012-02-28

    A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-{alpha} in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

  8. Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

    PubMed

    Yang, Yang; Cao, Min-Jie; Alcocer, Marcos; Liu, Qing-Mei; Fei, Dan-Xia; Mao, Hai-Yan; Liu, Guang-Ming

    2015-06-01

    Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability. PMID:25728640

  9. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  10. Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells.

    PubMed

    Kamei, Yuto; Tsang, Chi Kwan

    2003-08-01

    We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3 microg/ml MC14 in the presence of 1.25-100 ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10 microM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10 ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase-PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA-MAP kinases pathway and an adenylate cyclase-PKA pathway, in PC12D cells. PMID:12850058

  11. Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

    PubMed Central

    Zaitsevskaya-Carter, T; Cooper, J A

    1997-01-01

    A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

  12. β-arrestin drives MAP kinase signaling from clathrin-coated structures after GPCR dissociation

    PubMed Central

    Eichel, K.; Jullié, D.

    2016-01-01

    β-arrestins critically regulate G protein-coupled receptor (GPCR) signaling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G protein-independent signal via MAP kinase1–3. Despite enormous recent progress toward understanding biophysical aspects of arrestin function4,5, its cell biology remains relatively poorly understood. Two key tenets underlie the present dogma: (1) β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and (2) MAP kinase activation requires endocytosis of formed GPCR - β-arrestin complexes6–9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (Arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin and β-arrestin -dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signaling from CCSs. We propose a β-arrestin signaling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  13. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  14. Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development

    PubMed Central

    Ueda, Takeshi; Sasaki, Masato; Elia, Andrew J.; Chio, Iok In Christine; Hamada, Koichi; Fukunaga, Rikiro; Mak, Tak W.

    2010-01-01

    MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) are protein-serine/threonine kinases that are activated by ERK or p38 and phosphorylate eIF4E, which is involved in cap-dependent translation initiation. However, Mnk1/2 double knockout (Mnk-DKO) mice show normal cell growth and development despite an absence of eIF4E phosphorylation. Here we show that the tumorigenesis occurring in the Lck-Pten mouse model (referred to here as tPten−/− mice) can be suppressed by the loss of Mnk1/2. Phosphorylation of eIF4E was greatly enhanced in lymphomas of parental tPten−/− mice compared with lymphoid tissues of wild-type mice, but was totally absent in lymphomas of tPten−/−; Mnk-DKO mice. Notably, stable knockdown of Mnk1 in the human glioma cell line U87MG resulted in dramatically decreased tumor formation when these cells were injected into athymic nude mice. Our data demonstrate an oncogenic role for Mnk1/2 in tumor development, and highlight these molecules as potential anticancer drug targets that could be inactivated with minimal side effects. PMID:20679220

  15. Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes.

    PubMed

    Rime, H; Talbi, N; Popoff, M R; Suziedelis, K; Jessus, C; Ozon, R

    1998-12-15

    The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD. PMID:9882492

  16. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    PubMed Central

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments. PMID:9421504

  17. Phosphorylation of Bni4 by MAP kinases contributes to septum assembly during yeast cytokinesis.

    PubMed

    Pérez, Jacqueline; Arcones, Irene; Gómez, Alberto; Casquero, Verónica; Roncero, César

    2016-09-01

    Previous work has shown that the synthetic lethality of the slt2Δrim101Δ mutant results from a combination of factors, including improper functioning of the septum assembly machinery. Here, we identify new multicopy suppressors of this lethality including Kss1, Pcl1 and Sph1, none of which seems to be linked to the upregulation of chitin synthesis. Characterization of the suppression mediated by Kss1 showed that it is independent of the transcriptional response of the CWI signaling response, but efficiently restores the Bni4 localization defects produced by the absence of Slt2. Accordingly, Bni4 interacts physically with both kinases, and its levels of phosphorylation are reduced in the slt2Δ mutant but increased after Kss1 overexpression. Using an assay based on hypersensitive cells of the cdc10-11 mutant, we have pinpointed several MAP kinase phosphorylatable residues required for Bni4 function. Our results, together with a genetic correlation analysis, strongly support a functional model linking Slt2 MAP kinase and Pcl1, a Pho85 cyclin-dependent kinase, in septum assembly through Bni4. This model, based on the coordinated phosphorylation of Bni4 by both kinases, would be able to integrate cellular signals rapidly to maintain cell integrity during cytokinesis. PMID:27400980

  18. Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat.

    PubMed

    Takezawa, D

    1999-08-01

    Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments. Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase. The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens. BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA. A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein. Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation. The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract. These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat. Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts. PMID:10527417

  19. MAP kinase dynamics in response to pheromones in budding yeast.

    PubMed

    van Drogen, F; Stucke, V M; Jorritsma, G; Peter, M

    2001-12-01

    Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood. During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus. PMID:11781566

  20. Possible involvement of AMP-activated protein kinase in PGE1-induced synthesis of osteoprotegerin in osteoblasts

    PubMed Central

    KAINUMA, SHINGO; OTSUKA, TAKANOBU; KUROYANAGI, GEN; YAMAMOTO, NAOHIRO; MATSUSHIMA-NISHIWAKI, RIE; KOZAWA, OSAMU; TOKUDA, HARUHIKO

    2016-01-01

    AMP-activated protein kinase (AMPK) is firmly established as a central regulator of cellular energy homeostasis. We have previously reported that prostaglandin E1 (PGE1) stimulates the synthesis of osteoprotegerin through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. The present study investigated the involvement of AMPK in PGE1-induced osteoprotegerin synthesis in MC3T3-E1 cells. The levels of osteoprotegerin were measured using an enzyme-linked immunosorbent assay, while the phosphorylation of AMPK, acetyl-CoA carboxylase, p38 MAP kinase and SAPK/JNK were analyzed by western blotting. In addition, the mRNA expression levels of osteoprotegerin were determined by a reverse transcription-quantitative polymerase chain reaction. It was revealed that PGE1 significantly induced the phosphorylation of the α and β subunits of AMPK in a time-dependent manner (P<0.05). In addition, acetyl-CoA carboxylase, a direct substrate of AMPK, was significantly phosphorylated by PGE1 (P<0.05). Compound C, an AMPK inhibitor, was revealed to suppress the phosphorylation of acetyl-CoA carboxylase, which significantly reduced the release and mRNA expression levels of PGE1-stimulated osteoprotegerin (P<0.05). However, the PGE1-induced phosphorylation of p38 MAP kinase and SAPK/JNK were not affected by compound C. The results of the present study indicated that AMPK may positively regulate PGE1-stimulated osteoprotegerin synthesis in osteoblasts; thus providing novel insight into the regulatory mechanisms underlying bone metabolism. PMID:27168848

  1. Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    van den Berghe, N; Ouwens, D M; Maassen, J A; van Mackelenbergh, M G; Sips, H C; Krans, H M

    1994-01-01

    The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport. Images PMID:7511205

  2. Horse chestnut extract induces contraction force generation in fibroblasts through activation of Rho/Rho kinase.

    PubMed

    Fujimura, Tsutomu; Moriwaki, Shigeru; Hotta, Mitsuyuki; Kitahara, Takashi; Takema, Yoshinori

    2006-06-01

    Contraction forces generated by non-muscle cells such as fibroblasts play important roles in determining cell morphology, vasoconstriction, and/or wound healing. However, few factors that induce cell contraction forces are known, such as lysophosphatidic acid and thrombin. Our study analyzed various plant extracts for ingredients that induce generation of cell contraction forces in fibroblasts populating collagen gels. We found that an extract of Horse chestnut (Aesculus hippocastanum) is able to induce such contraction forces in fibroblasts. The involvement of actin polymerization and stress fiber formation in the force generation was suggested by inhibition of this effect by cytochalasin D and by Rhodamine phalloidin. Rho kinase inhibitors (Y27632 and HA1077) and a Rho inhibitor (exoenzyme C3) significantly inhibited the force generation induced by the Horse chestnut extract. H7, which inhibits Rho kinase as well as other protein kinases, also significantly inhibited induction of force generation. However, inhibitors of other protein kinases such as myosin light chain kinase (ML-9), protein kinase C (Calphostin), protein kinase A (KT5720), and tyrosine kinase (Genistein, Herbimycin A) had no effect on force generation induced by Horse chestnut extract. These results suggest that the Horse chestnut extract induces generation of contraction forces in fibroblasts through stress fiber formation followed by activation of Rho protein and Rho kinase but not myosin light chain kinase or other protein kinases. PMID:16754996

  3. The Upregulation of PI3K/Akt and MAP Kinase Pathways is Associated with Resistance of Microtubule-Targeting Drugs in Prostate Cancer.

    PubMed

    Liu, Zhi; Zhu, Guangjing; Getzenberg, Robert H; Veltri, Robert W

    2015-07-01

    Resistance is a significant limitation to the effectiveness of cancer therapies. The PI3K/Akt and MAP kinase pathways play important roles in a variety of normal cellular processes and tumorigenesis. This study is designed to explore the relationship of these signaling pathways with multidrug resistance in prostate cancer (PCa). The PI3K/Akt and MAP kinase pathways were investigated utilizing paclitaxel resistant DU145-TxR PCa cells and their parental non-resistant DU145 cells to determine their relationship with resistance to paclitaxel and other anticancer drugs. Our results demonstrate that the PI3K/Akt and MAP kinase pathways are upregulated in DU145-TxR cells compared to the DU145 cells. Inactivating these pathways using the PI3K/Akt pathway inhibitor LY294002 or the MAP kinase pathway inhibitor PD98059 renders the DU145-TxR cells more sensitive to paclitaxel. We investigated the effects of these inhibitors on other anticancer drugs including docetaxel, vinblastine, doxorubicin, 10-Hydroxycamptothecin (10-HCPT) and cisplatin and find that both inhibitors induces DU145-TxR cells to be more sensitive only to the microtubule-targeting drugs (paclitaxel, docetaxel and vinblastine). Furthermore, the treatment with these inhibitors induces cleaved-PARP production in DU145-TxR cells, suggesting that apoptosis induction might be one of the mechanisms for the reversal of drug resistance. In conclusion, the PI3K/Akt and MAP kinase pathways are associated with resistance to multiple chemotherapeutic drugs. Inactivating these pathways renders these PCa cells more sensitive to microtubule-targeting drugs such as paclitaxel, docetaxel and vinblastine. Combination therapies with novel inhibitors of these two signaling pathways potentially represents a more effective treatment for drug resistant PCa. PMID:25640606

  4. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ

    PubMed Central

    Aoto, Phillip C.; Martin, Bryan T.; Wright, Peter E.

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways. PMID:27353957

  5. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ.

    PubMed

    Aoto, Phillip C; Martin, Bryan T; Wright, Peter E

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways. PMID:27353957

  6. The Arabidopsis MAP kinase kinase 7: A crosstalk point between Auxin signaling and defense responses?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-pathogen interaction induces a complex host response that coordinates various signaling pathways through multiple signal molecules. Besides the well-documented signal molecules salicylic acid (SA), ethylene and jasmonic acid, auxin is emerging as an important player in this response. We recent...

  7. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  8. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis.

    PubMed

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W-Y; Puga, Alvaro; Xia, Ying

    2015-08-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1(+/-) embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  9. HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    PubMed Central

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

    2014-01-01

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of

  10. MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade.

    PubMed

    Ebert, Peter J R; Cheung, Jeanne; Yang, Yagai; McNamara, Erin; Hong, Rebecca; Moskalenko, Marina; Gould, Stephen E; Maecker, Heather; Irving, Bryan A; Kim, Jeong M; Belvin, Marcia; Mellman, Ira

    2016-03-15

    Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy. PMID:26944201

  11. Serum- and glucocorticoid-inducible kinases in microglia.

    PubMed

    Inoue, Koichi; Sakuma, Eisuke; Morimoto, Hiroyuki; Asai, Hayato; Koide, Yoshinori; Leng, Tiandong; Wada, Ikuo; Xiong, Zhi-Gang; Ueki, Takatoshi

    2016-09-01

    Microglia are derived from myelogenous cells and contribute to immunological and inflammatory responses in central nervous system. They play important roles not only in infectious diseases and inflammation after stroke, but also in psychiatric diseases such as schizophrenia. While recent studies suggest the significances of serum- and glucocorticoid-inducible kinases (SGKs) in other immune cells such as macrophages, T cells and dendritic cells, their role in microglia remains unknown. Here we, for the first time, report that SGK1 and SGK3 are expressed in multiple microglial cell lines. An SGK inhibitor, gsk650394, inhibits cell viability. In addition, lipopolysaccharide-induced expression of inflammatory regulators iNOS and TNFα was enhanced by gsk650394. Furthermore, translocation of NF-κB was enhanced by gsk650394. Taken together, these findings suggest that SGKs may play an important role in regulating microglial viability and inflammatory responses. PMID:27457803

  12. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    SciTech Connect

    Menschikowski, Mario; Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  13. Role of serum- and glucocorticoid-inducible kinases in stroke.

    PubMed

    Inoue, Koichi; Leng, Tiandong; Yang, Tao; Zeng, Zhao; Ueki, Takatoshi; Xiong, Zhi-Gang

    2016-07-01

    Increased expression of serum- and glucocorticoid-inducible kinase 1 (SGK1) can be induced by stress and growth factors in mammals, and plays an important role in cancer, diabetes, and hypertension. A recent work suggested that SGK1 activity restores damage in a stroke model. To further investigate the role of SGKs in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome in vivo and neurotoxicity in vitro. Infarct volumes were compared in adult mice with middle cerebral artery occlusion, followed by 24 h reperfusion, in the absence or presence of SGK inhibitors. Neurotoxicity assay, electrophysiological recording, and fluorescence Ca(2+) imaging were carried out using cultured cortical neurons to evaluate the underlying mechanisms. Contrary to our expectation, infarct volume by stroke decreased significantly when SGK inhibitor, gsk650394, or EMD638683, was administrated 30 min before middle cerebral artery occlusion under normal and diabetic conditions. SGK inhibitors reduced neurotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors, a leading factor responsible for cell death in stroke. SGK inhibitors also ameliorated Ca(2+) increase and peak amplitude of NMDA current in cultured neurons. In addition, SGK inhibitor gsk650394 decreased phosphorylation of Nedd4-2 and inhibited voltage-gated sodium currents. These observations suggest that SGK activity exacerbates stroke damage and that SGK inhibitors may be useful candidates for therapeutic intervention. To investigate the role of serum- and glucocorticoid-inducible kinases (SGKs) in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome. Infarct volumes induced by middle cerebral artery occlusion were decreased significantly by SGK inhibitors. The inhibitors also reduced glutamate toxicity, at least partly, by attenuation of NMDA and voltage-gated sodium currents. Thus, SGK inhibition attenuates stroke damage. PMID:27123541

  14. Loss of Raf Kinase Inhibitory Protein Induces Radioresistance in Prostate Cancer

    SciTech Connect

    Woods Ignatoski, Kathleen M.; Grewal, Navdeep K.; Markwart, Sonja M.; Vellaichamy, Adaikkalam; Chinnaiyan, Arul M.; Yeung, Kam; Ray, Michael E.; Keller, Evan T.

    2008-09-01

    Purpose: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. Methods and Materials: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. Results: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. Conclusion: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

  15. Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix.

    PubMed

    Sperandio, S; Poksay, K; de Belle, I; Lafuente, M J; Liu, B; Nasir, J; Bredesen, D E

    2004-10-01

    Programmed cell death (pcd) may take the form of apoptotic or nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here, we report that paraptosis, an alternative, nonapoptotic cell death program that may be induced by the insulin-like growth factor I receptor (among other inducers), is mediated by mitogen-activated protein kinases (MAPKs) and inhibited by AIP-1/Alix. The inhibition by AIP-1/Alix is specific for paraptosis since apoptosis was not inhibited. Caspases were not activated in this paradigm, nor were caspase inhibitors effective in blocking cell death. However, insulin-like growth factor I receptor (IGFIR)-induced paraptosis was inhibited by MEK-2-specific inhibitors and by antisense oligonucleotides directed against c-jun N-terminal kinase-1 (JNK-1). These results suggest that IGFIR-induced paraptosis is mediated by MAPKs, and inhibited by AIP-1/Alix. PMID:15195070

  16. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes

    PubMed Central

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells. PMID:26406906

  17. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    PubMed

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells. PMID:26406906

  18. Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening.

    PubMed

    Gangwal, Rahul P; Das, Nihar R; Thanki, Kaushik; Damre, Mangesh V; Dhoke, Gaurao V; Sharma, Shyam S; Jain, Sanyog; Sangamwar, Abhay T

    2014-04-01

    The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co-crystallised inhibitors were used in the development and validation of ligand and structure based pharmacophore modeling approached. The validated pharmacophore was utilized in database screening to identify potential hits. After Lipinski's rule of five filter and molecular docking analysis, nineteen hits were purchased and selected for in vitro analysis. The virtual hits exhibited promising activity against tumor necrosis factor-α (TNF-α) with 23-98% inhibition at 10μM concentration. Out of these seven compounds has shown potent inhibitory activity against p38 MAP kinase with IC50 values ranging from 12.97 to 223.5nM. In addition, the toxicity study against HepG2 cells was also carried out to confirm the safety profile of identified virtual hits. PMID:24473068

  19. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

    PubMed Central

    Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

    2016-01-01

    Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

  20. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    PubMed Central

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  1. Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

    PubMed Central

    Whisenant, Thomas C.; Ho, David T.; Benz, Ryan W.; Rogers, Jeffrey S.; Kaake, Robyn M.; Gordon, Elizabeth A.; Huang, Lan; Baldi, Pierre; Bardwell, Lee

    2010-01-01

    In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates. PMID:20865152

  2. Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis

    PubMed Central

    Meng, Xiangzong; Zhou, Jinggeng; Tang, Jiao; Li, Bo; de Oliveira, Marcos V. V.; Chai, Jijie; He, Ping; Shan, Libo

    2016-01-01

    SUMMARY Abscission is a developmental process that enables plants to shed unwanted organs. In Arabidopsis, the floral organ abscission is regulated by a signaling pathway consisting of the peptide ligand IDA, the receptor-like kinases (RLKs) HAE and HSL2, and a downstream MAP kinase (MAPK) cascade. However, little is known about the molecular link between ligand-receptor pairs and intracellular signaling. Here, we report that the SERK family RLKs function redundantly in regulating floral organ abscission downstream of IDA and upstream of the MAPK cascade. IDA induces heterodimerization of HAE/HSL2 and SERKs, which transphosphorylate each other. The SERK3 residues mediating its interaction with the immune receptor FLS2 and the brassinosteroid receptor BRI1 are also required for IDA-induced HAE/HSL2-SERK3 interaction, suggesting SERKs serve as co-receptors of HAE/HSL2 in perceiving IDA. Thus, our study reveals the signaling activation mechanism in floral organ abscission by IDA-induced HAE/HSL2-SERK complex formation accompanied by transphosphorylation. PMID:26854226

  3. Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

    PubMed Central

    Li, Dechang; Liu, Ming S.; Ji, Baohua

    2015-01-01

    Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems. PMID:26244746

  4. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

    PubMed Central

    Lavoie, Hugo; Thevakumaran, Neroshan; Gavory, Gwenaëlle; Li, John; Padeganeh, Abbas; Guiral, Sébastien; Duchaine, Jean; Mao, Daniel Y. L.; Bouvier, Michel; Sicheri, Frank; Therrien, Marc

    2016-01-01

    RAF kinases play a prominent role in cancer. Their mode of activation is complex, but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and as a result undesirably stimulate RAS/ERK-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe BRET-based biosensors for the extended RAF family enabling the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization as well as for probing structural determinants of RAF dimerization in vivo. Our findings, which appear generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. PMID:23685672

  5. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

    DOE PAGESBeta

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, II, David G.; Smith, Richard D.; Glass, N. Louise; Heitman, Joseph

    2014-11-20

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

  6. Activation of the MAP Kinase Cascade by Exogenous Calcium-Sensing Receptor

    SciTech Connect

    Hobson, Susan A.; Wright, Jay W.; Lee, Fred; Mcneil, Scott; Bilderback, Tim R.; Rodland, Karin D.

    2003-02-01

    In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled Calcium-sensing Receptor (CaR), as expression of the non-functional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3' kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR-mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses.

  7. Oxidative-stress-induced afterdepolarizations and calmodulin kinase II signaling.

    PubMed

    Xie, Lai-Hua; Chen, Fuhua; Karagueuzian, Hrayr S; Weiss, James N

    2009-01-01

    In the heart, oxidative stress caused by exogenous H(2)O(2) has been shown to induce early afterdepolarizations (EADs) and triggered activity by impairing Na current (I(Na)) inactivation. Because H(2)O(2) activates Ca(2+)/calmodulin kinase (CaMK)II, which also impairs I(Na) inactivation and promotes EADs, we hypothesized that CaMKII activation may be an important factor in EADs caused by oxidative stress. Using the patch-clamp and intracellular Ca (Ca(i)) imaging in Fluo-4 AM-loaded rabbit ventricular myocytes, we found that exposure to H(2)O(2) (0.2 to 1 mmol/L) for 5 to 15 minutes consistently induced EADs that were suppressed by the I(Na) blocker tetrodotoxin (10 micromol/L), as well as the I(Ca,L) blocker nifedipine. H(2)O(2) enhanced both peak and late I(Ca,L), consistent with CaMKII-mediated facilitation. By prolonging the action potential plateau and increasing Ca influx via I(Ca,L), H(2)O(2)-induced EADs were also frequently followed by DADs in response to spontaneous (ie, non-I(Ca,L)-gated) sarcoplasmic reticulum Ca release after repolarization. The CaMKII inhibitor KN-93 (1 micromol/L; n=4), but not its inactive analog KN-92 (1 micromol/L, n=5), prevented H(2)O(2)-induced EADs and DADs, and the selective CaMKII peptide inhibitor AIP (autocamtide-2-related inhibitory peptide) (2 micromol/L) significantly delayed their onset. In conclusion, H(2)O(2)-induced afterdepolarizations depend on both impaired I(Na) inactivation to reduce repolarization reserve and enhancement of I(Ca,L) to reverse repolarization, which are both facilitated by CaMKII activation. Our observations support a link between increased oxidative stress, CaMKII activation, and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased hearts. PMID:19038865

  8. S6 Kinase Reflects and Regulates Ethanol-Induced Sedation

    PubMed Central

    Acevedo, Summer F.; Peru y Colón de Portugal, Raniero L.; Gonzalez, Dante A.; Rodan, Aylin R.

    2015-01-01

    Alcohol use disorders (AUDs) affect people at great individual and societal cost. Individuals at risk for AUDs are sensitive to alcohol's rewarding effects and/or resistant to its aversive and sedating effects. The molecular basis for these traits is poorly understood. Here, we show that p70 S6 kinase (S6k), acting downstream of the insulin receptor (InR) and the small GTPase Arf6, is a key mediator of ethanol-induced sedation in Drosophila. S6k signaling in the adult nervous system determines flies' sensitivity to sedation. Furthermore, S6k activity, measured via levels of phosphorylation (P-S6k), is a molecular marker for sedation and overall neuronal activity: P-S6k levels are decreased when neurons are silenced, as well as after acute ethanol sedation. Conversely, P-S6k levels rebound upon recovery from sedation and are increased when neuronal activity is enhanced. Reducing neural activity increases sensitivity to ethanol-induced sedation, whereas neuronal activation decreases ethanol sensitivity. These data suggest that ethanol has acute silencing effects on adult neuronal activity, which suppresses InR/Arf6/S6k signaling and results in behavioral sedation. In addition, we show that activity of InR/Arf6/S6k signaling determines flies' behavioral sensitivity to ethanol-induced sedation, highlighting this pathway in acute responses to ethanol. SIGNIFICANCE STATEMENT Genetic factors play a major role in the development of addiction. Identifying these genes and understanding their molecular mechanisms is a necessary first step in the development of targeted therapeutic intervention. Here, we show that signaling from the insulin receptor in Drosophila neurons determines flies' sensitivity to ethanol-induced sedation. We show that this signaling cascade includes the small GTPase Arf6 and S6 kinase (S6k). In addition, activity of S6k is regulated by acute ethanol exposure and by neuronal activity. S6k activity is therefore both an acute target of ethanol exposure and

  9. MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397.

    PubMed

    Hayashida, Tomoko; Wu, Ming-Hua; Pierce, Amy; Poncelet, Anne-Christine; Varga, John; Schnaper, H William

    2007-12-01

    The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production. PMID:18032789

  10. Nuclear factor kappa B-inducing kinase and Ikappa B kinase-alpha signal skeletal muscle cell differentiation.

    PubMed

    Canicio, J; Ruiz-Lozano, P; Carrasco, M; Palacin, M; Chien, K; Zorzano, A; Kaliman, P

    2001-06-01

    Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II. PMID:11279241

  11. Activation of MAP kinase pathways in Galleria mellonella infected with Bacillus thuringiensis.

    PubMed

    Wojda, Iwona; Koperwas, Konrad; Jakubowicz, Teresa

    2014-01-01

    We followed changes in the level of phospho-MAP kinases in the greater wax moth Galleria mellonella after infection with Bacillus thuringiensis. We observed an enhanced level of phosphorylated p38 and JNK in fat bodies of the infected larvae. In hemocytes, injection of B. thuringiensis caused the highest increase in phospho-JNK, however, all pathways were activated after aseptic injection. We report that Galleria mellonella larvae exposed to heat shock before infection showed an enhanced level of phosphorylated JNK in fat body. This finding is relevant in the light of our previous reports, which submit evidence that pre-shocked animals are more resistant to infection. PMID:24455757

  12. The p38 MAP kinase pathway modulates the hypoxia response and glutamate receptor trafficking in aging neurons

    PubMed Central

    Park, Eun Chan; Rongo, Christopher

    2016-01-01

    Neurons are sensitive to low oxygen (hypoxia) and employ a conserved pathway to combat its effects. Here, we show that p38 MAP Kinase (MAPK) modulates this hypoxia response pathway in C. elegans. Mutants lacking p38 MAPK components pmk-1 or sek-1 resemble mutants lacking the hypoxia response component and prolyl hydroxylase egl-9, with impaired subcellular localization of Mint orthologue LIN-10, internalization of glutamate receptor GLR-1, and depression of GLR-1-mediated behaviors. Loss of p38 MAPK impairs EGL-9 protein localization in neurons and activates the hypoxia-inducible transcription factor HIF-1, suggesting that p38 MAPK inhibits the hypoxia response pathway through EGL-9. As animals age, p38 MAPK levels decrease, resulting in GLR-1 internalization; this age-dependent downregulation can be prevented through either p38 MAPK overexpression or removal of CDK-5, an antagonizing kinase. Our findings demonstrate that p38 MAPK inhibits the hypoxia response pathway and determines how aging neurons respond to hypoxia through a novel mechanism. DOI: http://dx.doi.org/10.7554/eLife.12010.001 PMID:26731517

  13. Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

    PubMed Central

    Morfini, Gerardo A.; Bosco, Daryl A.; Brown, Hannah; Gatto, Rodolfo; Kaminska, Agnieszka; Song, Yuyu; Molla, Linda; Baker, Lisa; Marangoni, M. Natalia; Berth, Sarah; Tavassoli, Ehsan; Bagnato, Carolina; Tiwari, Ashutosh; Hayward, Lawrence J.; Pigino, Gustavo F.; Watterson, D. Martin; Huang, Chun-Fang; Banker, Gary; Brown, Robert H.; Brady, Scott T.

    2013-01-01

    Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS. PMID:23776455

  14. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    PubMed

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  15. Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion ▿

    PubMed Central

    Sharma, Nishi R.; Mani, Prashant; Nandwani, Neha; Mishra, Rajakishore; Rana, Ajay; Sarkar, Debi P.

    2010-01-01

    Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy. PMID:20164223

  16. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    PubMed

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

  17. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

    PubMed Central

    Logan, S K; Falasca, M; Hu, P; Schlessinger, J

    1997-01-01

    The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells. PMID:9315636

  18. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  19. Chemical Genetics Approach Reveals Importance of cAMP and MAP Kinase Signaling to Lipid and Carotenoid Biosynthesis in Microalgae.

    PubMed

    Choi, Yoon-E; Rhee, Jin-Kyu; Kim, Hyun-Soo; Ahn, Joon-Woo; Hwang, Hyemin; Yang, Ji-Won

    2015-05-01

    In this study, we attempted to understand signaling pathways behind lipid biosynthesis by employing a chemical genetics approach based on small molecule inhibitors. Specific signaling inhibitors of MAP kinase or modulators of cAMP signaling were selected to evaluate the functional roles of each of the key signaling pathways in three different microalgal species: Chlamydomonas reinhardtii, Chlorella vulgaris, and Haematococcus pluvialis. Our results clearly indicate that cAMP signaling pathways are indeed positively associated with microalgal lipid biosynthesis. In contrast, MAP kinase pathways in three microalgal species are all negatively implicated in both lipid and carotenoid biosynthesis. PMID:25563422

  20. Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene

    SciTech Connect

    Ohshima, Toshio; Nagle, J.W.; Brady, R.O.; Kozak, C.A.

    1995-08-10

    Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5{prime}-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5. 20 refs., 2 figs., 1 tab.

  1. Leishmania major MAP kinase 10 is protective against experimental L. major infection.

    PubMed

    Kumari, Sangeeta; Singh, Sushma; Saha, Bhaskar; Paliwal, Piyush Kumar

    2011-11-01

    Leishmania, a protozoan parasite that resides and replicates obligatorily within macrophages, inflicts a complex of severe diseases known as leishmaniasis. The diseases have significant socio-economic impact through gross disfiguration, morbidity and mortality worldwide. Despite these problems, an effective anti-leishmanial vaccine remains elusive. Herein, we have analyzed the immunogenicity and protective efficacy of L. major MAP kinase 10 (LmjMAPK10) against the challenge infection with the parasite. We observe significant protection against the infection by LmjMAPK10 priming of BALB/c mouse strain, a susceptible host. The resistance to the infection is generally associated with mixed Th1/Th2 responses to the infection following immunization with LmjMAPK10 DNA or protein or a combination of both DNA and protein. Therefore, LmjMAPK10 is a probable vaccine candidate against the infection. PMID:21527301

  2. Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases.

    PubMed

    Baker, Paul R S; Owen, John S; Nixon, Andrew B; Thomas, Leslie N; Wooten, Rhonda; Daniel, Larry W; O'Flaherty, Joseph T; Wykle, Robert L

    2002-10-21

    Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2). PMID:12379481

  3. Role(s) of IL-2 inducible T cell kinase and Bruton's tyrosine kinase in mast cell response to lipopolysaccharide.

    PubMed

    Huang, Weishan; August, Avery

    2016-06-01

    Mast cells play critical roles during immune responses to the bacterial endotoxin lipopolysaccharide (LPS) that can lead to fatal septic hypothermia [1], [2], [3]. IL-2 inducible T cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are non-receptor tyrosine kinases that act downstream of numerous receptors, and have been shown to modulate mast cell responses downstream of FcεRIα [4], however, their roles in regulating mast cell responses to endotoxic stimuli were unclear. We found that the absence of ITK and BTK alters the mast cell response to LPS, and leads to enhanced pro-inflammatory cytokine production by mast cells and more severe LPS-induced hypothermia in mice [5]. Here, we detail our investigation using microarray analysis to study the transcriptomic profiles of mast cell responses to LPS, and the roles of ITK and/or BTK expression in this process. Mouse whole genome array data of WT, Itk (-/-) , Btk (-/-) , and Itk (-/-)  Btk (-/-) bone marrow-derived mast cells (BMMCs) stimulated by PBS (control) or LPS for 1 h were used in our latest research article [5] and is available in the Gene Expression Omnibus under accession number GSE64287. PMID:27081634

  4. Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

    PubMed Central

    Sharma, Vidhu; Young, Lisa; Cavadas, Miguel; Owen, Kate

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.11414.001 PMID:26999821

  5. Treatment with Histone Deacetylase Inhibitor Attenuates MAP Kinase Mediated Liver Injury in a Lethal Model of Septic Shock1

    PubMed Central

    Finkelstein, Robert A.; Li, Yongqing; Liu, Baoling; Shuja, Fahad; Fukudome, Eugene; Velmahos, George C.; deMoya, Marc; Alam, Hasan B.

    2016-01-01

    Background Despite global efforts to improve the treatment of sepsis, it remains a leading cause of morbidity and mortality in intensive care units. We have previously shown that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, markedly improves survival in a murine model of lipopolysaccharide (LPS)-induced shock. SAHA has anti-inflammatory properties that have not been fully characterized. The liver plays an important role in the production of acute phase reactants involved in the inflammatory cascade and is also one of the major organs that can become dysfunctional in septic shock. The purpose of this study was to assess the effect of SAHA treatment on MAP kinases and associated inflammatory markers in murine liver after LPS-induced injury. Methods C57B1/6J mice were randomly divided into three groups: (A) experimental-given intraperitoneal (i.p.) SAHA (50 mg/kg) in dimethyl sulfoxide (DMSO) vehicle solution (n =12); (B) control-given vehicle only (n = 12), and; (C) sham-given no treatment (n = 7). Two hours later, experimental and control mice were injected with LPS (20 mg/kg, i.p.) and experimental mice received a second dose of SAHA. Livers were harvested at 3, 24, and 48 h for analysis of inflammatory markers using Western Blot, Polymerase Chain Reaction (PCR), and Enzyme-Linked Immunosorbent Assay (ELISA) techniques. Results After 3 h, the livers of animals treated with SAHA showed significantly (P < 0.05) decreased expression of the pro-inflammatory MAP kinases phosphorylated p38, phosphorylated ERK, myeloperoxidase and interleukin-6, and increased levels of the anti-inflammatory interleukin-10 compared with controls. Phospho-p38 expression remained low in the SAHA treated groups at 24 and 48 h. Conclusion Administration of SAHA is associated with attenuation of MAPK activation and alteration of inflammatory and anti-inflammatory markers in murine liver after a lethal LPS insult. The suppression of MAPK activity is rapid (within 3 h

  6. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  7. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    PubMed

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  8. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers

    PubMed Central

    Adderley, Shaquria P.; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O.

    2015-01-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  9. Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC α and βII in Fetal Rat Cerebral Cortex Cultured Neuron

    PubMed Central

    Hasegawa, Jun; Takekoshi, Susumu; Nagata, Hidetaka; Osamura, R. Yoshiyuki; Suzuki, Toshiyasu

    2006-01-01

    Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral ­cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the ­localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0

  10. Histone Hyperacetylation Up-regulates Protein Kinase Cδ in Dopaminergic Neurons to Induce Cell Death

    PubMed Central

    Jin, Huajun; Kanthasamy, Arthi; Harischandra, Dilshan S.; Kondru, Naveen; Ghosh, Anamitra; Panicker, Nikhil; Anantharam, Vellareddy; Rana, Ajay; Kanthasamy, Anumantha G.

    2014-01-01

    The oxidative stress-sensitive protein kinase Cδ (PKCδ) has been implicated in dopaminergic neuronal cell death. However, little is known about the epigenetic mechanisms regulating PKCδ expression in neurons. Here, we report a novel mechanism by which the PKCδ gene can be regulated by histone acetylation. Treatment with histone deacetylase (HDAC) inhibitor sodium butyrate (NaBu) induced PKCδ expression in cultured neurons, brain slices, and animal models. Several other HDAC inhibitors also mimicked NaBu. The chromatin immunoprecipitation analysis revealed that hyperacetylation of histone H4 by NaBu is associated with the PKCδ promoter. Deletion analysis of the PKCδ promoter mapped the NaBu-responsive element to an 81-bp minimal promoter region. Detailed mutagenesis studies within this region revealed that four GC boxes conferred hyperacetylation-induced PKCδ promoter activation. Cotransfection experiments and Sp inhibitor studies demonstrated that Sp1, Sp3, and Sp4 regulated NaBu-induced PKCδ up-regulation. However, NaBu did not alter the DNA binding activities of Sp proteins or their expression. Interestingly, a one-hybrid analysis revealed that NaBu enhanced transcriptional activity of Sp1/Sp3. Overexpression of the p300/cAMP-response element-binding protein-binding protein (CBP) potentiated the NaBu-mediated transactivation potential of Sp1/Sp3, but expressing several HDACs attenuated this effect, suggesting that p300/CBP and HDACs act as coactivators or corepressors in histone acetylation-induced PKCδ up-regulation. Finally, using genetic and pharmacological approaches, we showed that NaBu up-regulation of PKCδ sensitizes neurons to cell death in a human dopaminergic cell model and brain slice cultures. Together, these results indicate that histone acetylation regulates PKCδ expression to augment nigrostriatal dopaminergic cell death, which could contribute to the progressive neuropathogenesis of Parkinson disease. PMID:25342743

  11. Targeting Apoptosis Signalling Kinase-1 (ASK-1) Does Not Prevent the Development of Neuropathy in Streptozotocin-Induced Diabetic Mice

    PubMed Central

    Newton, Victoria L.; Ali, Sumia; Duddy, Graham; Whitmarsh, Alan J.; Gardiner, Natalie J.

    2014-01-01

    Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy. PMID:25329046

  12. Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors

    PubMed Central

    Bretteville, Alexis; Marcouiller, François; Julien, Carl; El Khoury, Noura B.; Petry, Franck R.; Poitras, Isabelle; Mouginot, Didier; Lévesque, Georges; Hébert, Sébastien S.; Planel, Emmanuel

    2012-01-01

    Tau hyperphosphorylation is one hallmark of Alzheimer's disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors. PMID:22761989

  13. MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

    SciTech Connect

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala; Glas, Rickard

    2010-08-27

    Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  14. 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

  15. Role of p38 MAP Kinase Signal Transduction in Solid Tumors

    PubMed Central

    Pal, Mintu; Koul, Sweaty

    2013-01-01

    Mitogen-activated protein kinases (MAPKs) mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the main subgroups, the p38 MAP kinases, has been implicated in a wide range of complex biologic processes, such as cell proliferation, cell differentiation, cell death, cell migration, and invasion. Dysregulation of p38 MAPK levels in patients are associated with advanced stages and short survival in cancer patients (e.g., prostate, breast, bladder, liver, and lung cancer). p38 MAPK plays a dual role as a regulator of cell death, and it can either mediate cell survival or cell death depending not only on the type of stimulus but also in a cell type specific manner. In addition to modulating cell survival, an essential role of p38 MAPK in modulation of cell migration and invasion offers a distinct opportunity to target this pathway with respect to tumor metastasis. The specific function of p38 MAPK appears to depend not only on the cell type but also on the stimuli and/or the isoform that is activated. p38 MAPK signaling pathway is activated in response to diverse stimuli and mediates its function by components downstream of p38. Extrapolation of the knowledge gained from laboratory findings is essential to address the clinical significance of p38 MAPK signaling pathways. The goal of this review is to provide an overview on recent progress made in defining the functions of p38 MAPK pathways with respect to solid tumor biology and generate testable hypothesis with respect to the role of p38 MAPK as an attractive target for intervention of solid tumors. PMID:24349632

  16. Phosphorylation of MAP kinase-like proteins mediate the response of the halotolerant alga Dunaliella viridis to hypertonic shock.

    PubMed

    Jiménez, Carlos; Berl, Tomas; Rivard, Christopher J; Edelstein, Charles L; Capasso, Juan M

    2004-02-01

    The microalga Dunaliella viridis has the ability to adapt to a variety of environmental stresses including osmotic and thermal shocks, UV irradiation and nitrogen starvation. Lacking a rigid cell wall, Dunaliella provides an excellent model to study stress signaling in eukaryotic unicellular organisms. When exposed to hyperosmotic stress, UV irradiation or high temperature, a 57-kDa protein is recognized by antibodies specific to mammalian p38, to its yeast homologue Hog1, and to the phospho-p38 MAP kinase motif. This 57-kDa protein appears to be both up-regulated and phosphorylated. Three other proteins (50, 45, 43 kDa) were transiently phosphorylated under stress conditions as detected with an antibody specific to the mammalian phospho c-Jun N-terminal kinase (JNK) motif. Treatment with specific inhibitors of p38 MAP kinase (SB203580) and JNK (SP600125) activities markedly impaired the adaptation of Dunaliella to osmotic stress. From an evolutionary standpoint, these data strongly suggest that MAP kinase signaling pathways, other than ERK, were already operating in the common ancestor of plant and animal kingdoms, probably as early as 1400 million years ago. PMID:14741745

  17. A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

    PubMed Central

    Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

    2010-01-01

    MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

  18. Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase.

    PubMed Central

    Jacobs, D; Beitel, G J; Clark, S G; Horvitz, H R; Kornfeld, K

    1998-01-01

    Genetic analysis of lin-1 loss-of-function mutations suggests that lin-1 controls multiple cell-fate decisions during Caenorhabditis elegans development and is negatively regulated by a conserved receptor tyrosine kinase-Ras-ERK mitogen-activated protein (MAP) kinase signal transduction pathway. LIN-1 protein contains an ETS domain and presumably regulates transcription. We identified and characterized six gain-of-function mutations that define a new class of lin-1 allele. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting this region is required for negative regulation. The C terminus of LIN-1 was a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, our results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, that is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases. PMID:9691039

  19. Genetic variation in insulin-induced kinase signaling

    PubMed Central

    Wang, Isabel Xiaorong; Ramrattan, Girish; Cheung, Vivian G

    2015-01-01

    Individual differences in sensitivity to insulin contribute to disease susceptibility including diabetes and metabolic syndrome. Cellular responses to insulin are well studied. However, which steps in these response pathways differ across individuals remains largely unknown. Such knowledge is needed to guide more precise therapeutic interventions. Here, we studied insulin response and found extensive individual variation in the activation of key signaling factors, including ERK whose induction differs by more than 20-fold among our subjects. This variation in kinase activity is propagated to differences in downstream gene expression response to insulin. By genetic analysis, we identified cis-acting DNA variants that influence signaling response, which in turn affects downstream changes in gene expression and cellular phenotypes, such as protein translation and cell proliferation. These findings show that polymorphic differences in signal transduction contribute to individual variation in insulin response, and suggest kinase modulators as promising therapeutics for diseases characterized by insulin resistance. PMID:26202599

  20. Nitric Oxide Affects ERK Signaling through Down-Regulation of MAP Kinase Phosphatase Levels during Larval Development of the Ascidian Ciona intestinalis

    PubMed Central

    Palumbo, Anna

    2014-01-01

    In the ascidian Ciona intestinalis larval development and metamorphosis require a complex interplay of events, including nitric oxide (NO) production, MAP kinases (ERK, JNK) and caspase-3 activation. We have previously shown that NO levels affect the rate of metamorphosis, regulate caspase activity and promote an oxidative stress pathway, resulting in protein nitration. Here, we report that NO down-regulates MAP kinase phosphatases (mkps) expression affecting positively ERK signaling. By pharmacological approach, we observed that the reduction of endogenous NO levels caused a decrease of ERK phosphorylation, whereas increasing levels of NO induced ERK activation. We have also identified the ERK gene network affected by NO, including mpk1, mpk3 and some key developmental genes by quantitative gene expression analysis. We demonstrate that NO induces an ERK-independent down-regulation of mkp1 and mkp3, responsible for maintaining the ERK phosphorylation levels necessary for transcription of key metamorphic genes, such as the hormone receptor rev-erb and the van willebrand protein vwa1c. These results add new insights into the role played by NO during larval development and metamorphosis in Ciona, highlighting the cross-talk between different signaling pathways. PMID:25058405

  1. Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity

    PubMed Central

    Li, Bo; Jiang, Shan; Yu, Xiao; Cheng, Cheng; Chen, Sixue; Cheng, Yanbing; Yuan, Joshua S.; Jiang, Daohong; He, Ping; Shan, Libo

    2015-01-01

    Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression. PMID:25770109

  2. Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo.

    PubMed

    Jung, Sung Keun; Lee, Ki Won; Byun, Sanguine; Lee, Eun Jung; Kim, Jong-Eun; Bode, Ann M; Dong, Zigang; Lee, Hyong Joo

    2010-05-01

    Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1alpha expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70(S6K) in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin. PMID:20008033

  3. Mechanosensitive Kinases Regulate Stiffness-Induced Cardiomyocyte Maturation

    PubMed Central

    Young, Jennifer L.; Kretchmer, Kyle; Ondeck, Matthew G.; Zambon, Alexander C.; Engler, Adam J.

    2014-01-01

    Cells secrete and assemble extracellular matrix throughout development, giving rise to time-dependent, tissue-specific stiffness. Mimicking myocardial matrix stiffening, i.e. ~10-fold increase over 1 week, with a hydrogel system enhances myofibrillar organization of embryonic cardiomyocytes compared to static hydrogels, and thus we sought to identify specific mechanosensitive proteins involved. Expression and/or phosphorylation state of 309 unique protein kinases were examined in embryonic cardiomyocytes plated on either dynamically stiffening or static mature myocardial stiffness hydrogels. Gene ontology analysis of these kinases identified cardiogenic pathways that exhibited time-dependent up-regulation on dynamic versus static matrices, including PI3K/AKT and p38 MAPK, while GSK3β, a known antagonist of cardiomyocyte maturation, was down-regulated. Additionally, inhibiting GSK3β on static matrices improved spontaneous contraction and myofibril organization, while inhibiting agonist AKT on dynamic matrices reduced myofibril organization and spontaneous contraction, confirming its role in mechanically-driven maturation. Together, these data indicate that mechanically-driven maturation is at least partially achieved via active mechanosensing at focal adhesions, affecting expression and phosphorylation of a variety of protein kinases important to cardiomyogenesis. PMID:25236849

  4. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    PubMed

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear. PMID:25589570

  5. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors

    PubMed Central

    Riehl, Brandon D.; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-01-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear. PMID:25589570

  6. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    SciTech Connect

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  7. [Application of Kohonen Self-Organizing Feature Maps in QSAR of human ADMET and kinase data sets].

    PubMed

    Hegymegi-Barakonyi, Bálint; Orfi, László; Kéri, György; Kövesdi, István

    2013-01-01

    QSAR predictions have been proven very useful in a large number of studies for drug design, such as kinase inhibitor design as targets for cancer therapy, however the overall predictability often remains unsatisfactory. To improve predictability of ADMET features and kinase inhibitory data, we present a new method using Kohonen's Self-Organizing Feature Map (SOFM) to cluster molecules based on explanatory variables (X) and separate dissimilar ones. We calculated SOFM clusters for a large number of molecules with human ADMET and kinase inhibitory data, and we showed that chemically similar molecules were in the same SOFM cluster, and within such clusters the QSAR models had significantly better predictability. We used also target variables (Y, e.g. ADMET) jointly with X variables to create a novel type of clustering. With our method, cells of loosely coupled XY data could be identified and separated into different model building sets. PMID:24575660

  8. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    PubMed

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  9. Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism

    PubMed Central

    Sissons, James; Kim, Kwang Sik; Stins, Monique; Jayasekera, Samantha; Alsam, Selwa; Khan, Naveed Ahmed

    2005-01-01

    Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase. PMID:15845472

  10. Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

    PubMed

    Yamaguchi, Hiroto; Miwa, Yukiko; Kasa, Miyuki; Kitano, Ken; Amano, Mutsuki; Kaibuchi, Kozo; Hakoshima, Toshio

    2006-09-01

    Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability. PMID:16891330

  11. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    PubMed

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  12. ErbB2, EphrinB1, Src Kinase and PTPN13 Signaling Complex Regulates MAP Kinase Signaling in Human Cancers

    PubMed Central

    Vermeer, Paola D.; Bell, Megan; Lee, Kimberly; Vermeer, Daniel W.; Wieking, Byrant G.; Bilal, Erhan; Bhanot, Gyan; Drapkin, Ronny I.; Ganesan, Shridar; Klingelhutz, Aloysius J.; Hendriks, Wiljan J.; Lee, John H.

    2012-01-01

    In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression. PMID:22279592

  13. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

    PubMed Central

    D’Souza, Serena A.; Rajendran, Luckshika; Bagg, Rachel; van Pel, Derek M.; Moshiri, Houtan; Roy, Peter J.

    2016-01-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  14. A C. elegans p38 MAP kinase pathway mutant protects from dopamine, methamphetamine, and MDMA toxicity

    PubMed Central

    Schreiber, Matthew A.; McIntire, Steven L.

    2011-01-01

    Biogenic amine systems are damaged by amphetamine abuse and in Parkinson's disease. The mechanisms mediating this damage are of high importance because of the public health impact of these problems. Here we have taken advantage of the C. elegans nematode model system to investigate genetic modifiers of biogenic amine toxicity. In a forward genetic screen, we identified a mutant resistant to the toxic effects of dopamine. This mutant was also resistant to toxic doses of methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). In addition, this mutation conferred resistance to 6-hydroxydopamine damage to dopaminergic neurons in a Parkinson's disease model. Resistance was due to a mutation in the nsy-1 gene, orthologous to the mammalian ASK-1 MAPKKK. NSY-1 is in the highly conserved p38 MAP kinase pathway, which plays a crucial role in C. elegans innate immunity, suggesting that this pathway may play a role in biogenic amine toxicity system damage due to amphetamines and in the pathogenesis of Parkinson's disease in higher organisms. PMID:21565252

  15. Microglial Signaling in Chronic Pain with a Special Focus on Caspase 6, p38 MAP Kinase, and Sex Dependence.

    PubMed

    Berta, T; Qadri, Y J; Chen, G; Ji, R R

    2016-09-01

    Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6-a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia-is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some

  16. Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases, Which Control Stomata Development in Arabidopsis thaliana*

    PubMed Central

    Khan, Mamoona; Rozhon, Wilfried; Bigeard, Jean; Pflieger, Delphine; Husar, Sigrid; Pitzschke, Andrea; Teige, Markus; Jonak, Claudia; Hirt, Heribert; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module. PMID:23341468

  17. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

    PubMed Central

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

  18. Saucerneol F, a New Lignan Isolated from Saururus chinensis, Attenuates Degranulation via Phospholipase Cγ 1 Inhibition and Eicosanoid Generation by Suppressing MAP Kinases in Mast Cells.

    PubMed

    Lu, Yue; Son, Jong-Keun; Chang, Hyeun Wook

    2012-11-01

    During our on-going studies to identify bioactive compounds in medicinal herbs, we found that saucerneol F (SF), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis), showed in vitro anti-inflammatory activity. In this study, we examined the effects of SF on the generation of 5-lipoxygenase (5-LO) dependent leukotriene C4 (LTC4), cyclooxygenase-2 (COX-2) dependent prostaglandin D2 (PGD2), and on phospholipase Cγ1 (PLCγ1)-mediated degranulation in SCF-induced mouse bone marrow-derived mast cells (BMMCs). SF inhibited eicosanoid (PGD2 and LTC4) generation and degranulation dose-dependently. To identify the molecular mechanisms underlying the inhibition of eicosanoid generation and degranulation by SF, we examined the effects of SF on the phosphorylation of PLCγ1, intracellular Ca(2+) influx, the translocation of cytosolic phospholipase A2 (cPLA2) and 5-LO, and on the phosphorylation of MAP kinases (MAPKs). SF was found to reduce intracellular Ca(2+) influx by inhibiting PLCγ1 phosphorylation and suppressing the nuclear translocations of cPLA2 and 5-LO via the phosphorylations of MAPKs, including extracellular signal-regulated protein kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Taken together, these results suggest that SF may be useful for regulating mast cell-mediated inflammatory responses by inhibiting degranulation and eicosanoid generation. PMID:24009845

  19. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    PubMed

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  20. Overexpression of the MAP kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mitogen-activated protein kinases (MAPK) signaling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signaling in plants, a MAPK cDNA clone, OsMAPK33 was isolated from rice. The gene is mainly induced by ...

  1. Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.

    PubMed Central

    Nahas, N; Molski, T F; Fernandez, G A; Sha'afi, R I

    1996-01-01

    The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine

  2. CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19

    SciTech Connect

    Mock, B.A.; McBride, O.W.; Kozak, C.A.

    1995-05-20

    Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic {alpha}-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses. 17 refs., 2 figs., 1 tab.

  3. AICAR reduces the collagen-stimulated secretion of PDGF-AB and release of soluble CD40 ligand from human platelets: Suppression of HSP27 phosphorylation via p44/p42 MAP kinase

    PubMed Central

    Tsujimoto, Masanori; Tokuda, Haruhiko; Kuroyanagi, Gen; Yamamoto, Naohiro; Kainuma, Shingo; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Iida, Yuko; Kojima, Akiko; Sawada, Shigenobu; Doi, Tomoaki; Enomoto, Yukiko; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    We have previously reported that collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase in human platelets is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble cluster of differentiation 40 ligand (sCD40L). Adenosine monophosphate-activated protein kinase (AMPK), which is known to regulate energy homeostasis, has a crucial role as an energy sensor in various eukaryotic cells. The present study investigated the effects of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (AICAR), which is an activator of AMPK, on the collagen-induced activation of human platelets. It was demonstrated that AICAR dose-dependently reduced collagen-stimulated platelet aggregation up to 1.0 µM. Analysis of the size of platelet aggregates demonstrated that AICAR decreased the ratio of large aggregates (50–70 µm), whereas the ratio of small aggregates (9–25 µm) was increased by AICAR administration. AICAR markedly attenuated the phosphorylation levels of p44/p42 MAP kinase and HSP27, which are induced by collagen. Furthermore, AICAR significantly decreased the secretion of PDGF-AB and the collagen-induced release of sCD40L. These results indicated that AICAR-activated AMPK may reduce the secretion of PDGF-AB and the collagen-induced release of sCD40L by inhibiting HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

  4. AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.

    PubMed

    Zhu, Xiaoqing; Dahlmans, Vivian; Thali, Ramon; Preisinger, Christian; Viollet, Benoit; Voncken, J Willem; Neumann, Dietbert

    2016-08-12

    AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser(353) phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser(353) phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser(353) phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo. PMID:27413184

  5. The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors.

    PubMed

    Sun, Yongjun; Chen, You; Zhan, Liying; Zhang, Linan; Hu, Jie; Gao, Zibin

    2016-04-01

    Protein tyrosine phosphorylation is one of the primary modes of regulation of N-methyl-d-aspartate (NMDA) receptors. The non-receptor tyrosine kinases are one of the two types of protein tyrosine kinases that are involved in this process. The overactivation of NMDA receptors is a primary reason for neuron death following cerebral ischemia. Many studies have illustrated the important role of non-receptor tyrosine kinases in ischemia insults. This review introduces the roles of Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 in the excitotoxicity induced by the overactivation of NMDA receptors following cerebral ischemia. PMID:26540220

  6. The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

    PubMed Central

    Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

    2014-01-01

    OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

  7. Interleukin-4-induced transcriptional activation by stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6.

    PubMed

    Pesu, M; Takaluoma, K; Aittomäki, S; Lagerstedt, A; Saksela, K; Kovanen, P E; Silvennoinen, O

    2000-01-15

    Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3'-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway. (Blood. 2000;95:494-502) PMID:10627454

  8. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    ERIC Educational Resources Information Center

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  9. Cicletanine stimulates eNOS phosphorylation and NO production via Akt and MAP kinase/Erk signaling in sinusoidal endothelial cells.

    PubMed

    Liu, Songling; Rockey, Don C

    2013-07-15

    The function of the endothelial isoform of nitric oxide synthase (eNOS) and production of nitric oxide (NO) is altered in a number of disease states. Pharmacological approaches to enhancing NO synthesis and thus perhaps endothelial function could have substantial benefits in patients. We analyzed the effect of cicletanine, a synthetic pyridine with potent vasodilatory characteristics, on eNOS function and NO production in normal (liver) and injured rat sinusoidal endothelial cells, and we studied the effect of cicletanine-induced NO on stellate cell contraction and portal pressure in an in vivo model of liver injury. Sinusoidal endothelial cells were isolated from normal and injured rat livers. After exposure to cicletanine, eNOS phosphorylation, NO synthesis, and the signaling pathway regulating eNOS activation were measured. Cicletanine led to an increase in eNOS (Ser¹¹⁷⁷) phosphorylation, cytochrome c reductase activity, L-arginine conversion to L-citrulline, as well as NO production. The mechanism of the effect of cicletanine appeared to be via the protein kinase B (Akt) and MAP kinase/Erk signaling pathways. Additionally, cicletanine improved NO synthesis in injured sinusoidal endothelial cells. NO production induced by cicletanine in sinusoidal endothelial cells increased protein kinase G (PKG) activity as well as relaxation of stellate cells. Finally, administration of cicletanine to mice with portal hypertension induced by bile duct ligation led to reduction of portal pressure. The data indicate that cicletanine might improve eNOS activity in injured sinusoidal endothelial cells and likely activates hepatic stellate cell NO/PKG signaling. It raises the possibility that cicletanine could improve intrahepatic vascular function in portal hypertensive patients. PMID:23639812

  10. Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

    PubMed

    Polychronakos, C; Piscina, R; Fantus, I G

    1989-01-01

    Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia. PMID:2909378

  11. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

    PubMed Central

    Paul, A; Pendreigh, R H; Plevin, R

    1995-01-01

    1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. Images Figure 2 PMID:7533621

  12. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  13. Sorafenib induces apoptosis in HL60 cells by inhibiting Src kinase-mediated STAT3 phosphorylation.

    PubMed

    Zhao, Wei; Zhang, Tao; Qu, Bingqian; Wu, Xingxin; Zhu, Xu; Meng, Fanyu; Gu, Yanhong; Shu, Yongqian; Shen, Yan; Sun, Yang; Xu, Qiang

    2011-01-01

    Signal transducer and activator of transcription 3 (STAT3) is constitutively active in approximately 50% of acute myeloid leukemia (AML) cases and mediates multiple cellular processes including cell resistance to apoptosis. Inhibition of constitutively active STAT3 has been shown to induce AML cell apoptosis. Our aim was to ascertain if sorafenib, a multikinase inhibitor, may also inhibit STAT3 signaling and, therefore, be efficacious for AML. We found that sorafenib inhibited proliferation and induced apoptosis in human AML cell line (HL60) cells. In addition, sorafenib exposure reduced constitutive STAT3 phosphorylation in HL60 cells and repressed STAT3 DNA-binding activity and Mcl-1 and Bcl-2 expression. Similar results were obtained with the Src kinase inhibitor I, suggesting that sorafenib suppresses STAT3 phosphorylation by inhibiting Src-kinase activity. Furthermore, significant inhibition of Src kinase activity by sorafenib was observed in the kinase assay. In addition, Src could be co-immunoprecipitated with STAT3, and the phosphorylation of STAT3 was significantly inhibited by sorafenib only in cell lines in which phosphorylated Src is highly expressed. Taken together, our study indicates that sorafenib blocks Src kinase-mediated STAT3 phosphorylation and decreases the expression of apoptosis regulatory proteins Mcl-1 and Bcl-2, which are associated with increased apoptosis in HL60 cells. These findings provide a rationale for the treatment of human AML. PMID:20881478

  14. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells

    PubMed Central

    Rafiq, Rather A.; Quadri, Afnan; Nazir, Lone A.; Peerzada, Kaiser; Ganai, Bashir A.; Tasduq, Sheikh A.

    2015-01-01

    Ultraviolet (UV) radiation–induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  15. A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells.

    PubMed

    Rafiq, Rather A; Quadri, Afnan; Nazir, Lone A; Peerzada, Kaiser; Ganai, Bashir A; Tasduq, Sheikh A

    2015-01-01

    Ultraviolet (UV) radiation-induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future. PMID:26148186

  16. Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

    SciTech Connect

    Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

    2009-12-15

    Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

  17. Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

    PubMed

    Yin, Ziyi; Tang, Wei; Wang, Jingzhen; Liu, Xinyu; Yang, Lina; Gao, Chuyun; Zhang, Jinlong; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2016-06-01

    In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae. PMID:27193947

  18. MAP Kinase Phosphatase 1 (MKP-1/DUSP1) is Neuroprotective in Huntington’s Disease Via Additive Effects of JNK and p38 Inhibition

    PubMed Central

    Taylor, David M.; Moser, Roger; Régulier, Etienne; Breuillaud, Lionel; Dixon, Meredith; Beesen, Ayshe Ana; Elliston, Linda; Silva Santos, Mariana de Fatima; Kim, Jinho; Jones, Lesley; Goldstein, Darlene R.; Ferrante, Robert J.; Luthi-Carter, Ruth

    2013-01-01

    We previously demonstrated that sodium butyrate is neuroprotective in Huntington’s disease (HD) mice and that this therapeutic effect is associated with increased expression of mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1/DUSP1). Here we show that enhancing MKP-1 expression is sufficient to achieve neuroprotection in lentiviral models of HD. Wild-type MKP-1 overexpression inhibited apoptosis in primary striatal neurons exposed to an N-terminal fragment of polyglutamine-expanded huntingtin (Htt171-82Q), blocking caspase-3 activation and significantly reducing neuronal cell death. This neuroprotective effect of MKP-1 was demonstrated to be dependent on its enzymatic activity, being ablated by mutation of its phosphatase domain and being attributed to inhibition of specific MAP kinases (MAPKs). Overexpression of MKP-1 prevented the polyglutamine-expanded huntingtin-induced activation of c-Jun N-terminal kinases (JNKs) and p38 MAPKs, whereas extracellular signal-regulated kinase 1/2 (ERK1/2) activation was not altered by either polyglutamine-expanded Htt or MKP-1. Moreover, mutants of MKP-1 that selectively prevented p38 or JNK binding confirmed the important dual contributions of p38 and JNK regulation to MKP-1-mediated neuroprotection. These results demonstrate additive effects of p38 and JNK MAPK inhibition by MKP-1 without consequence to ERK activation in this striatal neuron-based paradigm. MKP-1 also provided neuroprotection in vivo in a lentiviral model of HD neuropathology in rat striatum. Taken together, these data extend previous evidence that JNK- and p38-mediated pathways contribute to HD pathogenesis and, importantly, show that therapies simultaneously inhibiting both JNK and p38 signalling pathways may lead to improved neuroprotective outcomes. PMID:23392662

  19. Degradation of the interferon-induced 68,000-M(r) protein kinase by poliovirus requires RNA.

    PubMed Central

    Black, T L; Barber, G N; Katze, M G

    1993-01-01

    Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its M(r) of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L. Black, B. Safer, A. Hovanessian, and M. G. Katze, J. Virol. 63:2244-2251, 1989). In the present study we investigated the mechanisms by which P68 degradation occurred. To do this we used an in vitro degradation assay which faithfully reproduced the in vivo events. Although viral gene expression was required for P68 degradation, the major poliovirus proteases, 2A and 3C, were found not to be directly involved with P68 proteolysis. However, the protease responsible for P68 degradation required divalent cations for maximal activity and probably has both an RNA and a protein component since trypsin and ribonuclease abrogated the activity. Despite this requirement for divalent cations and RNA, activation of the kinase was not required for proteolysis since a catalytically inactive P68 was still degraded. Mapping of P68 protease-sensitive sites by using in vitro translated truncation and deletion mutants revealed that sites required for degradation resided in the amino terminus and colocalized to dsRNA-binding domains. Finally, we found that preincubation of cell extracts with the synthetic dsRNA poly(I-C) largely prevented P68 proteolysis, providing additional evidence for the critical role of RNA. On the basis of these data, we present a hypothetical model depicting possible mechanisms of P68 degradation in poliovirus-infected cells. Images PMID:7678306

  20. AMP-activated protein kinase activation protects gastric epithelial cells from Helicobacter pylori-induced apoptosis.

    PubMed

    Lv, Guoqiang; Zhu, Huanhuan; Zhou, Feng; Lin, Zhou; Lin, Gang; Li, Chenwan

    2014-10-10

    Helicobacter pylori (H pylori), infecting half of the world's population, causes gastritis, duodenal and gastric ulcer, and gastric cancers. AMP-activated protein kinase (AMPK) is a highly conserved regulator of cellular energy and metabolism. Recent studies indicated an important role for AMPK in promoting cell survival. In this study, we discovered that H Pylori induced AMPK activation in transformed (GEC-1 line) and primary human gastric epithelial cells (GECs). Inhibition of H Pylori-stimulated AMPK kinase activity by AMPK inhibitor compound C exacerbated apoptosis in transformed and primary GECs. Meanwhile, downregulation of AMPK expression by targeted shRNAs promoted apoptosis in H pylori-infected GECs. In contrast, A-769662 and resveratrol, two known AMPK activators, or AMPKα1 over-expression, enhanced H Pylori-induced AMPK activation, and inhibited GEC apoptosis. Our data suggested that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) could be the upstream kinase for AMPK activation by H pylori. Partial depletion of TAK1 by shRNAs not only inhibited AMPK activation, but also suppressed survival of H pylori-infected GECs. Taken together, these results suggest that TAK1-dependent AMPK activation protects GECs from H pylori-Induced apoptosis. PMID:25229685

  1. Translational Control of Myelin Basic Protein Expression by ERK2 MAP Kinase Regulates Timely Remyelination in the Adult Brain

    PubMed Central

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine

    2015-01-01

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS. PMID:25995471

  2. Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain.

    PubMed

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine; Fyffe-Maricich, Sharyl L

    2015-05-20

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS. PMID:25995471

  3. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

    SciTech Connect

    Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, II, David G.; Smith, Richard D.; Glass, N. Louise; Heitman, Joseph

    2014-11-20

    Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal

  4. Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase

    PubMed Central

    Richter, Erik; Ventz, Katharina; Harms, Manuela; Mostertz, Jörg; Hochgräfe, Falko

    2016-01-01

    Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cyclin-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1) acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease. PMID:27066479

  5. Inhibition of Pim1 kinase, new therapeutic approach in virus-induced asthma exacerbations.

    PubMed

    Vries, Maaike de; Bedke, Nicole; Smithers, Natalie P; Loxham, Matthew; Howarth, Peter H; Nawijn, Martijn C; Davies, Donna E

    2016-03-01

    Therapeutic options to treat virus-induced asthma exacerbations are limited and urgently needed. Therefore, we tested Pim1 kinase as potential therapeutic target in human rhinovirus (HRV) infections. We hypothesised that inhibition of Pim1 kinase reduces HRV replication by augmenting the interferon-induced anti-viral response due to increased activity of the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway.Air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) from healthy individuals and moderate-to-severe asthmatic volunteers were infected with HRV-16 with or without a specific Pim1 inhibitor; viral replication and induction of anti-viral responses were measured using RT-qPCR. Viral titres were measured by 50% tissue culture infective dose and release of interferon-γ-induced protein 10 (IP-10) and RANTES protein assessed by ELISA. Phosphorylation of STAT-1 was determined using western blotting.Viral replication was reduced in ALI cultures of healthy and asthmatic PBECs treated with the Pim1 inhibitor. Using cultures from healthy donors, enhanced STAT-1 phosphorylation upon inhibition of Pim1 kinase activity resulted in increased mRNA expression of interferon-β, interleukin-29, IP-10 and RANTES 12 h after infection and increased protein levels of IP-10 and RANTES 24 h after infection.We have identified Pim1 kinase as novel target to reduce viral replication in ALI cultures of PBECs. This may open new avenues for therapeutic interventions in virus-induced asthma exacerbations. PMID:26869670

  6. Differences in Cell Death Induction by Phytophthora Elicitins Are Determined by Signal Components Downstream of MAP Kinase Kinase in Different Species of Nicotiana and Cultivars of Brassica rapa and Raphanus sativus[w

    PubMed Central

    Takemoto, Daigo; Hardham, Adrienne R.; Jones, David A.

    2005-01-01

    Elicitins are small, secreted proteins produced by species of the plant-pathogenic oomycete Phytophthora. They induce hypersensitive cell death in most Nicotiana species and in some cultivars of Brassica rapa and Raphanus sativus. In this study, two true-breeding Fast Cycling B. rapa lines were established that showed severe necrosis (line 7-R) or no visible response (line 18-NR) after treatment with elicitin. Unexpectedly, microscopic examination revealed localized cell death in line 18-NR plants, and expression levels of various defense-marker genes were comparable in both lines. These results suggested that both “responsive” and “nonresponsive” plants responded to elicitin but differed in the extent of the cell death response. Expression of a constitutively active form of Arabidopsis (Arabidopsis thaliana) MAP kinase kinase 4 (AtMEK4DD) also induced rapid development of confluent cell death in line 7-R, whereas line 18-NR showed no visible cell death. Similarly, elicitin-responsive Nicotiana species and R. sativus cultivars showed significantly stronger cell death responses following expression of AtMEK4DD compared with nonresponsive species/cultivars. Line 7-R also showed higher sensitivity to toxin-containing culture filtrates produced by Alternaria brassicicola, and toxin sensitivity cosegregated with elicitin responsiveness, suggesting that the downstream responses induced by elicitin and Alternaria toxin share factors that control the extent of cell death. Interestingly, elicitin responsiveness was shown to correlate with greater susceptibility to A. brassicicola (a necrotroph) in B. rapa but less susceptibility to Phytophthora nicotianae (a hemibiotroph) in Nicotiana, suggesting a more extensive cell death response could cause opposite effects on the outcomes of biotrophic versus necrotrophic plant-pathogen interactions. PMID:15980203

  7. Integrin-linked kinase: a hypoxia-induced anti-apoptotic factor exploited by cancer cells.

    PubMed

    Abboud, Elizabeth R; Coffelt, Seth B; Figueroa, Yanira G; Zwezdaryk, Kevin J; Nelson, Anne B; Sullivan, Deborah E; Morris, Cindy B; Tang, Yan; Beckman, Barbara S; Scandurro, Aline B

    2007-01-01

    Based on cDNA microarray results, integrin-linked kinase (ILK) emerged as an interesting candidate in hypoxia-mediated survival mechanisms employed by cancer cells. This notion was confirmed here by the following observations: the 5' promoter region of the ilk gene contains hypoxia responsive elements (HRE) that bind hypoxia-inducible factor (HIF) transcription factor complexes and drive HRE-luciferase gene expression in reporter assays; ILK protein and kinase activity are induced following hypoxia; downstream targets of ILK signaling are induced following hypoxia treatment; inhibition of ILK leads to increased apoptosis; and HIF and ILK are co-localized within human cancer tissues. The identification of ILK as a player in hypoxia survival signaling employed by cancer cells further validates ILK as a unique target for cancer therapy. PMID:17143519

  8. The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases

    PubMed Central

    Ozanne, James; Prescott, Alan R.; Clark, Kristopher

    2014-01-01

    Macrophages switch to an anti-inflammatory, ‘regulatory’-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of ‘regulatory’-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of ‘regulatory’-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of ‘regulatory’-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti

  9. Salt-Inducible Kinase 1 (SIK1) Is Induced by Gastrin and Inhibits Migration of Gastric Adenocarcinoma Cells

    PubMed Central

    Selvik, Linn-Karina M.; Rao, Shalini; Steigedal, Tonje S.; Haltbakk, Ildri; Misund, Kristine; Bruland, Torunn; Prestvik, Wenche S.; Lægreid, Astrid; Thommesen, Liv

    2014-01-01

    Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas. PMID:25384047

  10. Cadmium induces apoptosis in primary rat osteoblasts through caspase and mitogen-activated protein kinase pathways

    PubMed Central

    Zhao, Hongyan; Liu, Wei; Wang, Yi; Dai, Nannan; Gu, Jianhong; Yuan, Yan; Liu, Xuezhong; Bian, Jianchun

    2015-01-01

    Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs. PMID:26425111