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Sample records for mapk activation status

  1. Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK.

    PubMed

    Yang, Hui-Yu; Tatebayashi, Kazuo; Yamamoto, Katsuyoshi; Saito, Haruo

    2009-05-20

    The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG-specific Kss1 MAPK is activated by a combination of an O-glycosylation defect caused by disruption of the gene encoding the protein O-mannosyltransferase Pmt4, and an N-glycosylation defect induced by tunicamycin. The O-glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG-specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions. PMID:19369942

  2. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges.

    PubMed

    Gaestel, Matthias

    2015-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  3. Dehydrocorydaline promotes myogenic differentiation via p38 MAPK activation

    PubMed Central

    Yoo, Miran; Lee, Sang-Jin; Kim, Yong Kee; Seo, Dong-Wan; Baek, Nam-In; Ryu, Jae-Ha; Kang, Jong-Sun; Bae, Gyu-Un

    2016-01-01

    Muscle regeneration is a coordinated process that involves proliferation and differentiation of muscle progenitor cells. Activation of MyoD is a key event in myogenic differentiation, which is regulated by p38 mitogen-activated protein kinases (MAPK). In a screen of natural compounds for the enhancement of MyoD activity, dehydrocorydaline (DHC) from the Corydalis tuber was identified. Treatment of C2C12 myoblasts with DHC increased the expression levels of muscle-specific proteins, including MyoD, myogenin and myosin heavy chain. In addition, C2C12 myoblasts exhibited enhanced multinucleated myotube formation without any cytotoxicity. Treatment with DHC elevated p38 MAPK activation and the interaction of MyoD with an E protein, which is likely to result in activation of MyoD and promotion of myoblast differentiation. Furthermore, defects in differentiation-induced p38 MAPK activation and myoblast differentiation induced by depletion of the promyogenic receptor protein Cdo in C2C12 myoblasts were restored by DHC treatment. In conclusion, these results indicated that DHC stimulates p38 MAPK activation, which can enhance heterodimerization of MyoD and E proteins, thus resulting in MyoD activation and myoblast differentiation. These findings suggested that DHC may be considered a potential therapeutic compound for the improvement of muscle stem cell regenerative capacity in injured muscle. PMID:27573543

  4. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors

    PubMed Central

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W.; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D.; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J.; Modlin, Robert L.; Herschman, Harvey R.; Lo, Roger S.; McBride, William H.; Segura, Tatiana; Ribas, Antoni

    2016-01-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAFV600-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds. PMID:27476449

  5. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors.

    PubMed

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J; Modlin, Robert L; Herschman, Harvey R; Lo, Roger S; McBride, William H; Segura, Tatiana; Ribas, Antoni

    2016-08-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAF(V600)-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds.

  6. EhMAPK, the mitogen-activated protein kinase from Entamoeba histolytica is associated with cell survival.

    PubMed

    Ghosh, Anupama Sardar; Ray, Doel; Dutta, Suman; Raha, Sanghamitra

    2010-10-08

    Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.

  7. Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

    PubMed Central

    WIJAYAGUNAWARDANE, Missaka P. B.; HAMBRUCH, Nina; HAEGER, Jan-Dirk; PFARRER, Christiane

    2015-01-01

    Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival. PMID:26050642

  8. p38γ and p38δ Mitogen Activated Protein Kinases (MAPKs), New Stars in the MAPK Galaxy.

    PubMed

    Escós, Alejandra; Risco, Ana; Alsina-Beauchamp, Dayanira; Cuenda, Ana

    2016-01-01

    The protein kinases p38γ and p38δ belong to the p38 mitogen-activated protein kinase (MAPK) family. p38MAPK signaling controls many cellular processes and is one of the most conserved mechanisms in eukaryotes for the cellular response to environmental stress and inflammation. Although p38γ and p38δ are widely expressed, it is likely that they perform specific functions in different tissues. Their involvement in human pathologies such as inflammation-related diseases or cancer is starting to be uncovered. In this article we give a general overview and highlight recent advances made in defining the functions of p38γ and p38δ, focusing in innate immunity and inflammation. We consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases and cancer. PMID:27148533

  9. p38γ and p38δ Mitogen Activated Protein Kinases (MAPKs), New Stars in the MAPK Galaxy

    PubMed Central

    Escós, Alejandra; Risco, Ana; Alsina-Beauchamp, Dayanira; Cuenda, Ana

    2016-01-01

    The protein kinases p38γ and p38δ belong to the p38 mitogen-activated protein kinase (MAPK) family. p38MAPK signaling controls many cellular processes and is one of the most conserved mechanisms in eukaryotes for the cellular response to environmental stress and inflammation. Although p38γ and p38δ are widely expressed, it is likely that they perform specific functions in different tissues. Their involvement in human pathologies such as inflammation-related diseases or cancer is starting to be uncovered. In this article we give a general overview and highlight recent advances made in defining the functions of p38γ and p38δ, focusing in innate immunity and inflammation. We consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases and cancer. PMID:27148533

  10. Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus

    PubMed Central

    Neupane, Achal; Nepal, Madhav P; Benson, Benjamin V; MacArthur, Kenton J; Piya, Sarbottam

    2013-01-01

    Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution. PMID:24317362

  11. CpG- and LPS-activated MAPK signaling in in vitro cultured salmon (Salmo salar) mononuclear phagocytes.

    PubMed

    Iliev, Dimitar B; Hansen, Tom; Jørgensen, Sven Martin; Krasnov, Aleksei; Jørgensen, Jorunn B

    2013-10-01

    The Mitogen-activated protein kinases (MAPK) are involved in transmitting intracellular signals downstream of diverse cell surface receptors and mediate the response to ligands such as growth factors, hormones and cytokines. In addition, MAPK are critically involved in the innate immune response to pathogen-derived substances, commonly referred to as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS) and bacterial DNA rich in CpG dinucleotides. Currently, a great deal of knowledge is available about the involvement of MAPK in the innate immune response to PAMPs in mammals; however, little is known about the role of the different MAPK classes in the immune response to PAMPs in lower vertebrates. In the current study, p38 phosphorylation was induced by CpG oligonucleotides (ODNs) and LPS in primary salmon mononuclear phagocytes. Pre-treatment of the cells with a p38 inhibitor (SB203580) blocked the PAMP-induced p38 activity and suppressed the upregulation of most of the CpG- and LPS-induced transcripts highlighting the role of this kinase in the salmon innate immune response to PAMPs. In contrast to p38, the phosphorylation of extracellular signal-regulated kinase (ERK), a MAPK involved primarily in response to mitogens, was high in resting cells and, surprisingly, incubation with both CpG and control ODNs downregulated the phospho-ERK levels independently of p38 activation. The basal phospho-ERK level and the CpG-inducible p38 phosphorylation were greatly influenced by the length of in vitro incubation. The basal phospho-ERK level increased gradually throughout a 5-day culture period and was PI3K-dependent as demonstrated by its sensitivity to Wortmannin suggesting it is influenced by growth factors. Overall these data indicate that both basal and PAMP-induced activity of MAPKs might be greatly influenced by the differentiation status of salmon mononuclear phagocytes.

  12. Identification, Nomenclature, and Evolutionary Relationships of Mitogen-Activated Protein Kinase (MAPK) Genes in Soybean

    PubMed Central

    Neupane, Achal; Nepal, Madhav P.; Piya, Sarbottam; Subramanian, Senthil; Rohila, Jai S.; Reese, R. Neil; Benson, Benjamin V.

    2013-01-01

    Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution. PMID:24137047

  13. Mitogen-activated protein kinase phosphatase 1 negatively regulates MAPK signaling in mouse hypothalamus.

    PubMed

    Adachi, Koichi; Goto, Motomitsu; Onoue, Takeshi; Tsunekawa, Taku; Shibata, Miyuki; Hagimoto, Shigeru; Ito, Yoshihiro; Banno, Ryoichi; Suga, Hidetaka; Sugimura, Yoshihisa; Oiso, Yutaka; Arima, Hiroshi

    2014-05-21

    Mitogen-activated protein kinase phosphatase 1 (MKP-1) is shown to negatively regulate MAPK signaling in various peripheral tissues as well as the central nervous system such as cortex, striatum and hippocampus. In this study, we examined whether MKP-1 regulates MAPK signaling in the mouse hypothalamus. Intraperitoneal injection of TNFα significantly increased MKP-1 mRNA expression in paraventricular and arcuate nuclei in the hypothalamus. TNFα treatment induced increases in MKP-1 expression at both mRNA and protein levels, accompanied by the inactivation of MAPK signaling in mouse hypothalamic explants. Inhibition of MKP-1 by its inhibitor or siRNA increased MAPK activity in the explants. Our data indicate that MKP-1 negatively regulates MAPK signaling in the mouse hypothalamus.

  14. Cannabinoids inhibit the activation of ERK MAPK in PMA/Io-stimulated mouse splenocytes.

    PubMed

    Faubert Kaplan, Barbara L; Kaminski, Norbert E

    2003-10-01

    The mechanism of action of immune suppression by cannabinoids involves suppression of interleukin-2 (IL-2) production in phorbol ester plus calcium ionophore (PMA/Io)-stimulated lymphocytes. This decrease in IL-2 was due to inhibition of activator protein-1 (AP-1) and nuclear factor of activated T cells (NF-AT) transcription factors, both of which depend on proteins that are regulated by the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (ERK MAPK). Thus, the objective of the present study was to characterize the effects of cannabinoid compounds on ERK MAPK under conditions where IL-2 expression was suppressed. Using the MEK inhibitor PD098059 in order to assess the role of ERK MAPK in PMA/Io-stimulated splenocytes (SPLC), it was determined that IL-2 production and expression of c-fos and c-jun nuclear protein expression depended on activation of ERK MAPK. In response to PMA/Io, expression of nuclear phosphorylated ERK MAPK was rapidly induced, peaked at approximately 15 min, and was sustained for up to 240 min. Pretreatment with cannabinol (CBN) inhibited expression of phosphorylated ERK MAPK at several time points up to 240 min post cellular activation. Furthermore, WIN-55212-2, a synthetic cannabinoid, inhibited expression of phosphorylated ERK MAPK at 240 min post cellular activation. CBN did not induce activation of ERK MAPK in the absence of PMA/Io. Collectively, these studies suggest that cannabinoid-induced inhibition of IL-2 in PMA/Io-stimulated splenocytes might be due, in part, to inhibition of ERK MAPK activation.

  15. Role of amygdala MAPK activation on immobility behavior of forced swim rats.

    PubMed

    Huang, Tung-Yi; Lin, Chih-Hung

    2006-10-01

    The role of amygdala mitogen-activated protein kinase (MAPK) in rats during a forced swim test was investigated. The variation of amygdala MAPK level was studied in control rats and early-life maternally deprived rats. A forced swim test was carried out to estimate the immobility level. The data showed that the immobility time of rats that received maternal deprivation in early life was longer than that of control rats and Western blot analysis also showed that the amygdala phospho-MAPK level in maternally deprived rats was almost two times higher than in control rats. Intra-amygdala infusion of PD098059 or U0126, MEK inhibitors, suppressed immobility behavior during the forced swim test in both rats. Western blot analysis also showed that the amygdala MAPK activities in both rats infused with MEK inhibitors were also suppressed in parallel with expression of immobility behavior. The suppressed MAPK activities as well as the restoration of immobility time returned to the original level 48 h later. These results suggest that amygdala MAPK activation might play a role in the regulation of immobility behavior in rats during the forced swim test. Moreover, it could provide a hint that amygdala MAPK activation might be involved in the formation of depression-like behavior.

  16. Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

    PubMed

    Liu, Zhiyu; Wang, Bin; He, Ruijun; Zhao, Yanmei; Miao, Long

    2014-02-01

    In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

  17. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    SciTech Connect

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

  18. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

    PubMed Central

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-01-01

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

  19. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    DOE PAGES

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

  20. Immunosuppressant MPA Modulates Tight Junction through Epigenetic Activation of MLCK/MLC-2 Pathway via p38MAPK

    PubMed Central

    Khan, Niamat; Pantakani, D. V. Krishna; Binder, Lutz; Qasim, Muhammad; Asif, Abdul R.

    2015-01-01

    Background: Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) prescribed to prevent graft rejection in the organ transplanted patients, however, its use is also associated with adverse side effects like sporadic gastrointestinal (GI) disturbances. Recently, we reported the MPA induced tight junctions (TJs) deregulation which involves MLCK/MLC-2 pathway. Here, we investigated the global histone acetylation as well as gene-specific chromatin signature of several genes associated with TJs regulation in Caco-2 cells after MPA treatment. Results: The epigenetic analysis shows that MPA treatment increases the global histone acetylation levels as well as the enrichment for transcriptional active histone modification mark (H3K4me3) at promoter regions of p38MAPK, ATF-2, MLCK, and MLC-2. In contrast, the promoter region of occludin was enriched for transcriptional repressive histone modification mark (H3K27me3) after MPA treatment. In line with the chromatin status, MPA treatment increased the expression of p38MAPK, ATF-2, MLCK, and MLC-2 both at transcriptional and translational level, while occludin expression was negatively influenced. Interestingly, the MPA induced gene expression changes and functional properties of Caco-2 cells could be blocked by the inhibition of p38MAPK using a chemical inhibitor (SB203580). Conclusions: Collectively, our results highlight that MPA disrupts the structure of TJs via p38MAPK-dependent activation of MLCK/MLC-2 pathway that results in decreased integrity of Caco-2 monolayer. These results led us to suggest that p38MAPK-mediated lose integrity of epithelial monolayer could be the possible cause of GI disturbance (barrier dysfunction) in the intestine, leading to leaky style diarrhea observed in the organ-transplanted patients treated with MPA. PMID:26733876

  1. Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea.

    PubMed

    Iakovleva, Nadya V; Gorbushin, Alexander M; Storey, Kenneth B

    2006-09-01

    The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK. PMID:16533608

  2. Molecular Changes Involving MEK3-p38 MAPK Activation in Chronic Masticatory Myalgia.

    PubMed

    Meng, H; Gao, Y; Kang, Y F; Zhao, Y P; Yang, G J; Wang, Y; Cao, Y; Gan, Y H; Xie, Q F

    2016-09-01

    The exact mechanism underlying chronic masticatory myalgia (CMM), a conspicuous symptom in temporomandibular disorders, remains unclear. This investigation compared gene expression profiles between CMM patients and healthy subjects. Peripheral blood leukocytes were collected in 8 cases and 8 controls and subjected to whole genome microarray analyses. Data were analyzed with Gene Ontology and interactive pathways analyses. According to Gene Ontology analysis, categories such as ion transport, response to stimuli, and metabolic process were upregulated. The pathway analysis suggested overexpression of the mitogen-activated protein kinase (MAPK) pathway in CMM patients and to a higher degree in a pathway network. Overexpression of representative members of the MAPK pathway-including MAPK kinase 3 (MEK3), calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2), and growth arrest and DNA damage-inducible gamma (GADD45G)-was validated with real-time polymerase chain reaction. The upregulation of MEK3 was negatively correlated with the age of the CMM group. In the next step, the authors focused on MEK3, the gene that exhibited the greatest degree of differential expression, and its downstream target protein p38 MAPK. The results revealed upregulation of MEK3, as well as phosphorylated MEK3 and phosphorylated p38 MAPK, in CMM patients. These results provide a "fingerprint" for mechanistic studies of CMM in the future and highlight the importance of MEK3-p38 MAPK activation in CMM. PMID:27418173

  3. Reciprocal actions of NCAM and tPA via a Ras-dependent MAPK activation in rat hippocampal neurons.

    PubMed

    Son, Hyeon; Seuk Kim, Jin; Mogg Kim, Jung; Lee, Sang-Hun; Lee, Yong-Sung

    2002-10-25

    In an attempt to identify the functions of neural cell adhesion molecule (NCAM) and tissue plasminogen activator (tPA) in hippocampal synaptic plasticity, we investigated the relationship between the two molecules by focusing on mitogen-activated protein kinase (MAPK), an essential enzyme in this process. NCAM clustering in cultured hippocampal neurons transiently induced MAPK within 10min. Moreover, soluble NCAM also induced a Ras-dependent MAPK activation. Conversely, MAPK activation led to an increase in the expressions of all three isoforms of NCAM. Treatment of neurons with tPA and plasminogen induced a Ras-dependent MAPK activation and tPA-plasmin degradation of NCAM was mediated in a MAPK-dependent manner. Soluble NCAM transiently inhibited tPA mRNA expression levels in a MAPK-dependent manner, while stimulation of MAPK alone induced tPA reduction in cells. These results collectively indicate that NCAM and tPA reciprocally act as important regulators in the modulation of synaptic plasticity via a Ras-MAPK-involved signaling pathway. In turn, MAPK activation may cause tPA degradation or a decrease in expression to promote synaptic plasticity.

  4. Thrombin Enhances NGF-Mediated Neurite Extension via Increased and Sustained Activation of p44/42 MAPK and p38 MAPK

    PubMed Central

    Mufti, Rania E.; Sarker, Krishna; Jin, Yan; Fu, Songbin; Rosales, Jesusa L.; Lee, Ki-Young

    2014-01-01

    Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling. PMID:25061982

  5. Evaluating the Activity of the Filamentous Growth MAPK Pathway in Yeast

    PubMed Central

    Cullen, Paul J.

    2015-01-01

    Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signaling pathways that regulate diverse processes in eukaryotes. One such pathway regulates filamentous growth, a nutrient limitation response in budding yeast and other fungal species. This protocol describes three assays used to measure the activity of the filamentous growth pathway. First, western blotting for phosphorylated (activated) MAPKs (P~MAPKs; Slt2p, Kss1p, Fus3p, and Hog1p) provides a measure of MAPK activity in yeast and other fungal species. Second, the PGU1 gene is a transcriptional target of the filamentous growth pathway. Cells that undergo filamentous growth secrete Pgu1, an endopolygalacturonase that degrades the plant-specific polysaccharide pectin. We describe an assay that measures secreted pectinase activity, which reflects an active filamentous growth pathway. Finally, in yeast, two mucin-like glycoproteins, Msb2 and Flo11, regulate filamentous growth. Secretion of the processed and shed glycodomain of Msb2 is an indicator of MAPK activity. Flo11, the major adhesion molecule that controls filamentous growth and biofilm/mat formation, is also shed from cells. Detecting shed mucins with epitope-tagged versions of the proteins (secretion profiling) provides information about the regulation of filamentous growth across fungal species. PMID:25734070

  6. Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous-growth signalling pathway.

    PubMed

    Cook, J G; Bardwell, L; Thorner, J

    1997-11-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved signalling modules that regulate responses to diverse extracellular stimuli, developmental cues and environmental stresses. A MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is activated by an upstream protein kinase, such as Raf, Mos or a MAPKK kinase. Ste7, a MAPKK in the yeast Saccharomyces cerevisiae, is required for two developmental pathways: mating and invasive (filamentous) growth. Kss1 and Fus3, the MAPK targets of Ste7, are required for mating, but their role in invasive growth has been unclear. Because no other S. cerevisiae MAPK has been shown to function in invasive growth, it was proposed that Ste7 may have non-MAPK targets. We show instead that Kss1 is the principal target of Ste7 in the invasive-growth response in both haploids and diploids. We demonstrate further that Kss1 in its inactive form is a potent negative regulator of invasive growth. Ste7 acts to relieve this negative regulation by switching Kss1 from an inhibitor to an activator. These results indicate that this MAPK has a physiologically important function in its unactivated state. Comparison of normal and MAPK-deficient cells indicates that nitrogen starvation and activated Ras stimulate filamentous growth through both MAPK-independent and MAPK-dependent means. PMID:9363895

  7. Suppression of B-Raf(V600E) cancers by MAPK hyper-activation

    PubMed Central

    Eldad, Sophia; Smeir, Elia; Bar-Tana, Jacob

    2016-01-01

    B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells. PMID:26959890

  8. Suppression of B-Raf(V600E) cancers by MAPK hyper-activation.

    PubMed

    Atiq, Rawan; Hertz, Rachel; Eldad, Sophia; Smeir, Elia; Bar-Tana, Jacob

    2016-04-01

    B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells. PMID:26959890

  9. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    SciTech Connect

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa . E-mail: baekjh@snu.ac.kr

    2006-08-18

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.

  10. Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: Novel insights into diverse MAPK outcomes

    PubMed Central

    2011-01-01

    Background The yeast cell wall integrity mitogen-activated protein kinase (CWI-MAPK) pathway is the main regulator of adaptation responses to cell wall stress in yeast. Here, we adopt a genomic approach to shed light on two aspects that are only partially understood, namely, the characterization of the gene functional catalog associated with CWI pathway activation and the extent to which MAPK activation correlates with transcriptional outcomes. Results A systematic yeast mutant deletion library was screened for constitutive transcriptional activation of the CWI-related reporter gene MLP1. Monitoring phospho-Slt2/Mpk1 levels in the identified mutants revealed sixty-four deletants with high levels of phosphorylation of this MAPK, including mainly genes related to cell wall construction and morphogenesis, signaling, and those with unknown function. Phenotypic analysis of the last group of mutants suggests their involvement in cell wall homeostasis. A good correlation between levels of Slt2 phosphorylation and the magnitude of the transcriptional response was found in most cases. However, the expression of CWI pathway-related genes was enhanced in some mutants in the absence of significant Slt2 phosphorylation, despite the fact that functional MAPK signaling through the pathway was required. CWI pathway activation was associated to increased deposition of chitin in the cell wall - a known survival compensatory mechanism - in about 30% of the mutants identified. Conclusion We provide new insights into yeast genes related to the CWI pathway and into how the state of activation of the Slt2 MAPK leads to different outcomes, discovering the versatility of this kind of signaling pathways. These findings potentially have broad implications for understanding the functioning of other eukaryotic MAPKs. PMID:21810245

  11. Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons

    PubMed Central

    Ehrich, Jonathan M.; Messinger, Daniel I.; Knakal, Cerise R.; Kuhar, Jamie R.; Schattauer, Selena S.; Bruchas, Michael R.; Zweifel, Larry S.; Kieffer, Brigitte L.; Phillips, Paul E.M.

    2015-01-01

    The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR−/− mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood

  12. Dehydrocorydaline promotes myogenic differentiation via p38 MAPK activation.

    PubMed

    Yoo, Miran; Lee, Sang-Jin; Kim, Yong Kee; Seo, Dong-Wan; Baek, Nam-In; Ryu, Jae-Ha; Kang, Jong-Sun; Bae, Gyu-Un

    2016-10-01

    Muscle regeneration is a coordinated process that involves proliferation and differentiation of muscle progenitor cells. Activation of MyoD is a key event in myogenic differentiation, which is regulated by p38 mitogen‑activated protein kinases (MAPK). In a screen of natural compounds for the enhancement of MyoD activity, dehydrocorydaline (DHC) from the Corydalis tuber was identified. Treatment of C2C12 myoblasts with DHC increased the expression levels of muscle‑specific proteins, including MyoD, myogenin and myosin heavy chain. In addition, C2C12 myoblasts exhibited enhanced multinucleated myotube formation without any cytotoxicity. Treatment with DHC elevated p38 MAPK activation and the interaction of MyoD with an E protein, which is likely to result in activation of MyoD and promotion of myoblast differentiation. Furthermore, defects in differentiation‑induced p38 MAPK activation and myoblast differentiation induced by depletion of the promyogenic receptor protein Cdo in C2C12 myoblasts were restored by DHC treatment. In conclusion, these results indicated that DHC stimulates p38 MAPK activation, which can enhance heterodimerization of MyoD and E proteins, thus resulting in MyoD activation and myoblast differentiation. These findings suggested that DHC may be considered a potential therapeutic compound for the improvement of muscle stem cell regenerative capacity in injured muscle.

  13. Dehydrocorydaline promotes myogenic differentiation via p38 MAPK activation.

    PubMed

    Yoo, Miran; Lee, Sang-Jin; Kim, Yong Kee; Seo, Dong-Wan; Baek, Nam-In; Ryu, Jae-Ha; Kang, Jong-Sun; Bae, Gyu-Un

    2016-10-01

    Muscle regeneration is a coordinated process that involves proliferation and differentiation of muscle progenitor cells. Activation of MyoD is a key event in myogenic differentiation, which is regulated by p38 mitogen‑activated protein kinases (MAPK). In a screen of natural compounds for the enhancement of MyoD activity, dehydrocorydaline (DHC) from the Corydalis tuber was identified. Treatment of C2C12 myoblasts with DHC increased the expression levels of muscle‑specific proteins, including MyoD, myogenin and myosin heavy chain. In addition, C2C12 myoblasts exhibited enhanced multinucleated myotube formation without any cytotoxicity. Treatment with DHC elevated p38 MAPK activation and the interaction of MyoD with an E protein, which is likely to result in activation of MyoD and promotion of myoblast differentiation. Furthermore, defects in differentiation‑induced p38 MAPK activation and myoblast differentiation induced by depletion of the promyogenic receptor protein Cdo in C2C12 myoblasts were restored by DHC treatment. In conclusion, these results indicated that DHC stimulates p38 MAPK activation, which can enhance heterodimerization of MyoD and E proteins, thus resulting in MyoD activation and myoblast differentiation. These findings suggested that DHC may be considered a potential therapeutic compound for the improvement of muscle stem cell regenerative capacity in injured muscle. PMID:27573543

  14. [Studies on cell signaling immunomodulated murine peritoneal suppressor macrophages: LPS and PMA mediate the activation of RAF-1, MAPK p44 and MAPK p42 and p38 MAPK].

    PubMed

    Chang, Z L; Lin, M Q; Wang, M Z; Yao, Z

    1997-03-01

    Monocytes-macrophages which serve as host immune cells to kill pathogens can often be "activated" after exposing to viruses, bacteria, cytokines as well as chemical substances, However, it is paradoxical that highly activated macrophages can be induced to become the suppressor ones by live microbes, microbial products, tumor, and autoimmune disease, although the mechanism remains unknown. Our previous experimental studies have shown that immuno-suppressor activities of suppressor macrophages on T, B and NK cells can be prevented by the treatment with LPS or supernatant in vitro from mitogen-stimulated lymphocytes, while, at the same time, the tumoricidal activities of those macrophages can be kept or even enhanced following the same treatment. This phenomenon was then termed as "immune modulation" For the understanding of its mechanism, we are now undertaking signal transduction in modulated macrophages. Since mitogen-activated protein kinase (MAPK) is an integration point of different signal transduction pathways, its cascade and regulation of activation are being investigated extensively by the assay of electrophoresis mobility shift. Recent results suggested that interaction of ligand-receptor triggers protein tyrosine kinase(PTK) activation leading to Ras-GTP binding with Raf-1 to phosphorylate MAPK kinase (MAPKK), the specific activator of MAPK. It is reported that PKC-alpha can directly phosphorylate or activate Raf-1 in NIH3 T3 cells. Raf-1 (74 KDa), with an intrinsic serine (Ser)-threonine (The) kinase activity, becomes hyperphosphorylated after activation which can be followed by gel mobility shift test. It has also been shown that a variety of extracellular factors stimulate a pair of MAPK p44 and MAPK p42 of MAPK family members. A significant property of activation of ERK 1 and ERK 2 is the requirement for the phosphorylation of both Thr-183 and Tyr-185 (at TEY motif) within in its protein kinase subdomain VIII. More recently, two other MAPK subtypes, p

  15. EPAS1 trans-activation during hypoxia requires p42/p44 MAPK.

    PubMed

    Conrad, P W; Freeman, T L; Beitner-Johnson, D; Millhorn, D E

    1999-11-19

    Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-alpha/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-alpha (HIF1-alpha). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-alpha activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-induced trans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for the trans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation. PMID:10559262

  16. Pheromone-Induced Morphogenesis Improves Osmoadaptation Capacity by Activating the HOG MAPK Pathway**

    PubMed Central

    Baltanás, Rodrigo; Bush, Alan; Couto, Alicia; Durrieu, Lucía; Hohmann, Stefan; Colman-Lerner, Alejandro

    2013-01-01

    Environmental and internal conditions expose cells to a multiplicity of stimuli whose consequences are difficult to predict. Here, we investigate the response to mating pheromone of yeast cells adapted to high osmolarity. Events downstream of pheromone binding involve two mitogen-activated protein kinase (MAPK) cascades: the pheromone response (PR) and the cell-wall integrity response (CWI). Although these MAPK pathways share components with each and a third MAPK pathway, the high osmolarity response (HOG), they are normally only activated by distinct stimuli, a phenomenon called insulation. We found that in cells adapted to high osmolarity, PR activated the HOG pathway in a pheromone- and osmolarity- dependent manner. Activation of HOG by the PR was not due to loss of insulation, but rather a response to a reduction in internal osmolarity, which resulted from an increase in glycerol release caused by the PR. By analyzing single-cell time courses, we found that stimulation of HOG occurred in discrete bursts that coincided with the “shmooing” morphogenetic process. Activation required the polarisome, the cell wall integrity MAPK Slt2, and the aquaglyceroporin Fps1. HOG activation resulted in high glycerol turnover that improved adaptability to rapid changes in osmolarity. Our work shows how a differentiation signal can recruit a second, unrelated sensory pathway to enable responses to yeast to multiple stimuli. PMID:23612707

  17. Hog1 mitogen-activated protein kinase (MAPK) interrupts signal transduction between the Kss1 MAPK and the Tec1 transcription factor to maintain pathway specificity.

    PubMed

    Shock, Teresa R; Thompson, James; Yates, John R; Madhani, Hiten D

    2009-04-01

    In Saccharomyces cerevisiae, the mating, filamentous growth (FG), and high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) signaling pathways share components and yet mediate distinct responses to different extracellular signals. Cross talk is suppressed between the mating and FG pathways because mating signaling induces the destruction of the FG transcription factor Tec1. We show here that HOG pathway activation results in phosphorylation of the FG MAPK, Kss1, and the MAPKK, Ste7. However, FG transcription is not activated because HOG signaling prevents the activation of Tec1. In contrast to the mating pathway, we find that the mechanism involves the inhibition of DNA binding by Tec1 rather than its destruction. We also find that nuclear accumulation of Tec1 is not affected by HOG signaling. Inhibition by Hog1 is apparently indirect since it does not require any of the consensus S/TP MAPK phosphorylation sites on Tec1, its DNA-binding partner Ste12, or the associated regulators Dig1 or Dig2. It also does not require the consensus MAPK sites of the Ste11 activator Ste50, in contrast to a recent proposal for a role for negative feedback in specificity. Our results demonstrate that HOG signaling interrupts the FG pathway signal transduction between the phosphorylation of Kss1 and the activation of DNA binding by Tec1. PMID:19218425

  18. Differential Effects of E2 on MAPK Activity in the Brain and Heart of Aged Female Rats

    PubMed Central

    Shults, Cody L.; Rao, Yathindar S.; Pak, Toni R.

    2016-01-01

    Aging and the coincident loss of circulating estrogens at menopause lead to increased risks for neurological and cardiovascular pathologies. Clinical studies show that estrogen therapy (ET) can be beneficial in mitigating these negative effects, in both the brain and heart, when it is initiated shortly after the perimenopausal transition. However, this same therapy is detrimental when initiated >10 years postmenopause. Importantly, the molecular mechanisms underlying this age-related switch in ET efficacy are unknown. Estrogen receptors (ERs) mediate the neuroprotective and cardioprotective functions of estrogens by modulating gene transcription or, non-genomically, by activating second messenger signaling pathways, such as mitogen activated protein kinases (MAPK). These kinases are critical regulators of cell signaling pathways and have widespread downstream effects. Our hypothesis is that age and estrogen deprivation following menopause alters the expression and activation of the MAPK family members p38 and ERK in the brain and heart. To test this hypothesis, we used a surgically induced model of menopause in 18 month old rats through bilateral ovariectomy (OVX) followed by an acute dose of 17β-estradiol (E2) administered at varying time points post-OVX (1 week, 4 weeks, 8 weeks, or 12 weeks). Age and E2 treatment differentially regulated kinase activity in both the brain and heart, and the effects were also brain region specific. MAPK signaling plays an integral role in aging, and the aberrant regulation of those signaling pathways might be involved in age-related disorders. Clinical studies show benefits of ET during early menopause but detrimental effects later, which might be reflective of changes in kinase expression and activation status. PMID:27487271

  19. Differential Effects of E2 on MAPK Activity in the Brain and Heart of Aged Female Rats.

    PubMed

    Pinceti, Elena; Shults, Cody L; Rao, Yathindar S; Pak, Toni R

    2016-01-01

    Aging and the coincident loss of circulating estrogens at menopause lead to increased risks for neurological and cardiovascular pathologies. Clinical studies show that estrogen therapy (ET) can be beneficial in mitigating these negative effects, in both the brain and heart, when it is initiated shortly after the perimenopausal transition. However, this same therapy is detrimental when initiated >10 years postmenopause. Importantly, the molecular mechanisms underlying this age-related switch in ET efficacy are unknown. Estrogen receptors (ERs) mediate the neuroprotective and cardioprotective functions of estrogens by modulating gene transcription or, non-genomically, by activating second messenger signaling pathways, such as mitogen activated protein kinases (MAPK). These kinases are critical regulators of cell signaling pathways and have widespread downstream effects. Our hypothesis is that age and estrogen deprivation following menopause alters the expression and activation of the MAPK family members p38 and ERK in the brain and heart. To test this hypothesis, we used a surgically induced model of menopause in 18 month old rats through bilateral ovariectomy (OVX) followed by an acute dose of 17β-estradiol (E2) administered at varying time points post-OVX (1 week, 4 weeks, 8 weeks, or 12 weeks). Age and E2 treatment differentially regulated kinase activity in both the brain and heart, and the effects were also brain region specific. MAPK signaling plays an integral role in aging, and the aberrant regulation of those signaling pathways might be involved in age-related disorders. Clinical studies show benefits of ET during early menopause but detrimental effects later, which might be reflective of changes in kinase expression and activation status. PMID:27487271

  20. FGFR2c-mediated ERK-MAPK activity regulates coronal suture development.

    PubMed

    Pfaff, Miles J; Xue, Ke; Li, Li; Horowitz, Mark C; Steinbacher, Derek M; Eswarakumar, Jacob V P

    2016-07-15

    Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis. PMID:27034231

  1. Activation of MAPK Is Necessary for Long-Term Memory Consolidation Following Food-Reward Conditioning

    ERIC Educational Resources Information Center

    Ribeiro, Maria J.; Schofield, Michael G.; Kemenes, Ildiko; O'Shea, Michael; Kemenes, Gyorgy; Benjamin, Paul R.

    2005-01-01

    Although an important role for the mitogen-activated protein kinase (MAPK) has been established for memory consolidation in a variety of learning paradigms, it is not known if this pathway is also involved in appetitive classical conditioning. We address this question by using a single-trial food-reward conditioning paradigm in the freshwater…

  2. Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

    PubMed

    Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

    2014-01-01

    Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

  3. Brominated Flame Retardants, Tetrabromobisphenol A and Hexabromocyclododecane, Activate Mitogen-Activated Protein Kinases (MAPKs) in Human Natural Killer Cells

    PubMed Central

    Cato, Anita; Celada, Lindsay; Kibakaya, Esther Caroline; Simmons, Nadia; Whalen, Margaret M.

    2014-01-01

    NK cells provide a vital surveillance against virally infected cells, tumor cells, and antibody-coated cells through the release of cytolytic mediators and gamma interferon (IFN-γ). Hexabromocyclododecane (HBCD) is a brominated flame retardant used primarily in expanded (EPS) and extruded (XPS) polystyrene foams for thermal insulation in the building and construction industry. Tetrabromobisphenol A (TBBPA) is used both as a reactive and an additive flame retardant in a variety of materials. HBCD and TBBPA contaminate the environment and are found in human blood samples. In previous studies, we have shown that other environmental contaminants, such as the dibutyltin (DBT) and tributyltin (TBT), decrease NK lytic function by activating mitogen-activated protein kinases (MAPKs) in the NK cells. HBCD and TBBPA also interfere with NK cell(s) lytic function. The current study evaluates whether HBCD and/or TBBPA have the capacity to activate MAPKs and MAPK kinases (MAP2Ks). The effects of concentrations of HBCD and TBBPA that inhibited lytic function on the phosphorylation state and total levels of the MAPKs (p44/42, p38, and JNK) and the phosphorylation and total levels of the MAP2Ks (MEK1/2 and MKK3/6) were examined. Results indicate that exposure of human NK cells to 10-0.5 µM HBCD or TBBPA activate MAPKs and MAP2Ks. This HBCD and TBBPA-induced activation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contributes to the observed decreases in lytic function seen in NK cells exposed to HBCD and TBBPA. PMID:25341744

  4. MAPK specificity in the yeast pheromone response independent of transcriptional activation.

    PubMed

    Breitkreutz, A; Boucher, L; Tyers, M

    2001-08-21

    The mechanisms whereby different external cues stimulate the same mitogen-activated protein kinase (MAPK) cascade, yet trigger an appropriately distinct biological response, epitomize the conundrum of specificity in cell signaling. In yeast, shared upstream components of the mating pheromone and filamentous growth pathways activate two related MAPKs, Fus3 and Kss1, which in turn regulate programs of gene expression via the transcription factor Ste12. As fus3, but not kss1, strains are impaired for mating, Fus3 exhibits specificity for the pheromone response. To account for this specificity, it has been suggested that Fus3 physically occludes Kss1 from pheromone-activated signaling complexes, which are formed on the scaffold protein Ste5. However, we find that genome-wide expression profiles of pheromone-treated wild-type, fus3, and kss1 deletion strains are highly correlated for all induced genes and, further, that two catalytically inactive versions of Fus3 fail to abrogate the pheromone-induced transcriptional response. Consistently, Fus3 and Kss1 kinase activity is induced to an equivalent extent in pheromone-treated cells. In contrast, both in vivo and in an in vitro-reconstituted MAPK system, Fus3, but not Kss1, exhibits strong substrate selectivity toward Far1, a bifunctional protein required for polarization and G(1) arrest. This effect accounts for the failure to repress G(1)-S specific transcription in fus3 strains and, in part, explains the mating defect of such strains. MAPK specificity in the pheromone response evidently occurs primarily at the substrate level, as opposed to specific kinase activation by dedicated signaling complexes. PMID:11525741

  5. Neferine induces autophagy of human ovarian cancer cells via p38 MAPK/ JNK activation.

    PubMed

    Xu, Limei; Zhang, Xiyu; Li, Yinuo; Lu, Shuhua; Lu, Shan; Li, Jieyin; Wang, Yuqiong; Tian, Xiaoxue; Wei, Jian-Jun; Shao, Changshun; Liu, Zhaojian

    2016-07-01

    Ovarian cancer is the most lethal gynecological malignancy. Patients usually have poor prognosis because of late diagnosis, relapse, and chemoresistance. It is pressing to seek novel agent for the treatment of ovarian cancer. Neferine is a bisbenzylisoquinoline alkaloid isolated from the embryos of lotus (Nelumbo nucifera). In this study, we investigated the antitumor effect of neferine on ovarian cancer cells. We found that neferine exhibited growth-inhibitory effect on human ovarian cancer cells, whereas showing less cytotoxic to non-malignant fallopian tube epithelial cells. Furthermore, we demonstrated that neferine induced autophagy and inactivated the mTOR pathway. Finally, we found that both p38 MAPK and JNK signaling pathways were activated by neferine treatment and contributed to the induction of autophagy in ovarian cancer cells. In conclusion, our findings showed that neferine induced autophagy of human ovarian cancer cells via p38 MAPK/JNK activation. Neferine may be explored as a promising antitumoral agent in ovarian cancer. PMID:26738868

  6. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-09-02

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway.

  7. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-01-01

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway. PMID:27598116

  8. Ganoderma lucidum polysaccharide exerts anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

    PubMed

    Yang, Guohua; Yang, Lei; Zhuang, Yun; Qian, Xifeng; Shen, Yunfeng

    2016-01-01

    In this study, we investigated the anti-tumor activity both in vitro and in vivo of a polysaccharide obtained from Ganoderma lucidum on HL-60 acute myeloid leukemia cells, and focused on its targeting effect on mitogen-activated protein kinase (MAPK) pathways. It was found by the methods such as western blot and flow cytometry (FCM), that G. lucidum polysaccharide (GLP) blocked the extracellular signal-regulated kinase/MAPK signaling pathway, simultaneously activated p38 and JNK MAPK pathways, and therefore regulated their downstream genes and proteins, including p53, c-myc, c-fos, c-jun, Bcl-2, Bax, cleaved caspase-3 and cyclin D1. As a result, cycle arrest and apoptosis of HL-60 cells were induced. Therefore, GLP exerted anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

  9. Rice GDP dissociation inhibitor 3 inhibits OsMAPK2 activity through physical interaction.

    PubMed

    Heo, Jae Bok; Yi, Young Byung; Bahk, Jeong Dong

    2011-11-01

    GDP dissociation inhibitor (GDI) plays an essential role in regulating the state of bound nucleotides and subcellular localizations of Rab proteins. In our previous study, we showed that OsGDI3 facilitates the recycling of OsRab11 with a help of OsGAP1. In this study, we show that OsGDI3 complement the yeast sec19-1 mutant, a temperature-sensitive allele of the yeast GDI gene, suggesting that OsGDI3 is a functional ortholog of yeast GDI. To obtain further knowledge on the function of OsGDI3, candidate OsGDI3-interacting proteins were identified by yeast two-hybrid screens. OsMAPK2 is one of OsGDI3 interacting proteins from yeast two-hybrid screens and subject to further analysis. A kinase assay showed that the autophosphorylation activity of OsMAPK2 is inhibited by OsGDI3 in vitro. In addition, ectopic expressions of OsGDI3-in Arabidopsis cause reductions at the level of phosphorylated AtMPK in phosphorylation activity. Taken together, OsGDI3 functions as a negative regulator of OsMAPK2 through modulating its kinase activity.

  10. Impact of MAPK Pathway Activation in BRAF(V600) Melanoma on T Cell and Dendritic Cell Function.

    PubMed

    Ott, Patrick A; Bhardwaj, Nina

    2013-10-28

    Constitutive upregulation of the MAPK pathway by a BRAF(V600) mutation occurs in about half of melanomas. This leads to increased oncogenic properties such as tumor cell invasion, metastatic potential, and resistance to apoptosis. Blockade of the MAPK pathway with highly specific kinase inhibitors induces unprecedented tumor response rates in patients with advanced BRAF(V600) mutant melanoma. Immune checkpoint blockade with monoclonal antibodies targeting cytotoxic T-lymphocyte antigen 4 and programed death-1/PD-L1 has also demonstrated striking anti-tumor activity in patients with advanced melanoma. Tumor responses are likely limited by multiple additional layers of immune suppression in the tumor microenvironment. There is emerging preclinical and clinical evidence suggesting that MAPK inhibition has a beneficial effect on the immunosuppressive tumor microenvironment, providing a strong rationale for combined immunotherapy and MAPK pathway inhibition in melanoma. The T cell response has been the main focus in the studies reported to date. Since dendritic cells (DCs) are important in the induction of tumor-specific T cell responses, the impact of MAPK pathway activation in melanoma on DC function is critical for the melanoma directed immune response. BRAF(V600E) melanoma cells modulate DCs through the MAPK pathway because its blockade in melanoma cells can reverse suppression of DC function. As both MEK/BRAF inhibition and immune checkpoint blockade have recently taken center stage in the treatment of melanoma, a deeper understanding of how MAPK pathway inhibition affects the tumor immune response is needed.

  11. Leptin Promotes cPLA₂ Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade.

    PubMed

    Hsu, Pei-Sung; Wu, Chi-Sheng; Chang, Jia-Feng; Lin, Wei-Ning

    2015-11-18

    Hyperplasia or hypertrophy of adipose tissues plays a crucial role in obesity, which is accompanied by the release of leptin. Recently, obesity was determined to be associated with various pulmonary diseases including asthma, acute lung injury, and chronic obstructive pulmonary disease. However, how obesity contributes to pulmonary diseases and whether leptin directly regulates lung inflammation remains unclear. We used cell and animal models to study the mechanisms of leptin mediation of pulmonary inflammation. We found that leptin activated de novo synthesis of cytosolic phospholipase A₂-α (cPLA₂-α) in vitro in the lung alveolar type II cells, A549, and in vivo in ICR mice. Upregulated cPLA₂-α protein was attenuated by pretreatment with an OB-R blocking antibody, U0126, SB202190, SP600125, Bay11-7086, garcinol, and p300 siRNA, suggesting roles of p42/p44 MAPK, p38 MAPK, JNK1/2, NF-κB, and p300 in leptin effects. Leptin enhanced the activities of p42/p44 MAPK, p38 MAPK, JNK1/2, and p65 NF-κB in a time-dependent manner. Additional studies have suggested the participation of OB-R, p42/p44 MAPK, and JNK1/2 in leptin-increased p65 phosphorylation. Furthermore, p300 phosphorylation and histone H4 acetylation were reduced by blockage of OB-R, p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB in leptin-stimulated cells. Similarly, blockage of the MAPKs/NF-κB/p300 cascade significantly inhibited leptin-mediated cPLA₂-α mRNA expression. Our data as a whole showed that leptin contributed to lung cPLA₂-α expression through OB-R-dependent activation of the MAPKs/NF-κB/p300 cascade.

  12. A novel p38 mitogen activated protein kinase (MAPK) specific inhibitor suppresses respiratory syncytial virus and influenza A virus replication by inhibiting virus-induced p38 MAPK activation.

    PubMed

    Choi, Myung-Soo; Heo, Jinyuk; Yi, Chae-Min; Ban, Junsu; Lee, Noh-Jin; Lee, Na-Rae; Kim, Sang Won; Kim, Nam-Jung; Inn, Kyung-Soo

    2016-08-26

    Respiratory syncytial virus (RSV) and influenza A virus are leading causes of acute lower respiratory infectious disease. Respiratory diseases caused by RSV and influenza A virus result in serious economic burden and life-threatening disease for immunocompromised people. With the revelation that p38 mitogen-activated protein kinase (MAPK) activity in host cells is crucial for infection and replication of RSV and influenza A virus, inhibition of p38 MAPK activity has been suggested as a potential antiviral therapeutic strategy. However, the low selectivity and high toxicity of the p38 MAPK inhibitors necessitate the development of better inhibitors. Herein, we report the synthesis of a novel p38 MAPK inhibitor, NJK14047, with high kinase selectivity. In this work, it was demonstrated that NJK14047 inhibits RSV- and influenza A-mediated p38 MAPK activation in epithelial cells. Subsequently, NJK14047 treatment resulted in decreased viral replication and viral mRNA synthesis. In addition, secretion of interleukin-6 from infected cells was greatly diminished by NJK14047, suggesting that it can ameliorate immunopathological responses to RSV and influenza A. Collectively, the results suggest that NJK14047 has therapeutic potential to treat respiratory viral infection through the suppression of p38 MAPK activation, which is suggested to be an essential step for respiratory virus infection. PMID:27346133

  13. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation

    PubMed Central

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A. Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation. PMID:26262877

  14. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    PubMed

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  15. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids.

    PubMed

    Soares-Silva, Mercedes; Diniz, Flavia F; Gomes, Gabriela N; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host's MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  16. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids

    PubMed Central

    Soares-Silva, Mercedes; Diniz, Flavia F.; Gomes, Gabriela N.; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host’s MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  17. The activation of p38MAPK and JNK pathways in bovine herpesvirus 1 infected MDBK cells.

    PubMed

    Zhu, Liqian; Yuan, Chen; Huang, Liyuan; Ding, Xiuyan; Wang, Jianye; Zhang, Dong; Zhu, Guoqiang

    2016-01-01

    We have shown previously that BHV-1 infection activates Erk1/2 signaling. Here, we show that BHV-1 provoked an early-stage transient and late-stage sustained activation of JNK, p38MAPK and c-Jun signaling in MDBK cells. C-Jun phosphorylation was dependent on JNK. These early events were partially due to the viral entry process. Unexpectedly, reactive oxygen species were not involved in the later activation phase. Interestingly, only activated JNK facilitated the viral multiplication identified through both chemical inhibitor and siRNA. Collectively, this study provides insight into our understanding of early stages of BHV-1 infection. PMID:27590675

  18. Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

    PubMed Central

    Eberhard, Ralf; Stergiou, Lilli; Hofmann, E. Randal; Hofmann, Jen; Haenni, Simon; Teo, Youjin; Furger, André; Hengartner, Michael O.

    2013-01-01

    Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. PMID:24278030

  19. Involvement of p38 MAPK in the Anticancer Activity of Cultivated Cordyceps militaris.

    PubMed

    Chou, Shang-Min; Lai, Wan-Jung; Hong, Tzuwen; Tsai, Sheng-Hong; Chen, Yen-Hsun; Kao, Cheng-Hsiang; Chu, Richard; Shen, Tang-Long; Li, Tsai-Kun

    2015-01-01

    Cordyceps militaris is a traditional Chinese medicine frequently used for tonic and therapeutic purposes. Reports from our laboratory and others have demonstrated that extracts of the cultivated fruiting bodies of C. militaris (CM) exhibit a potent cytotoxic effect against many cancer cell lines, especially human leukemia cells. Here, we further investigated the underlying mechanism through which CM is cytotoxic to cancer cells. The CM-mediated induction of PARP cleavage and its related DNA damage signal (γH2AX) was diminished by caspase inhibitor I. In contrast, a ROS scavenger failed to prevent CM-mediated leukemia cell death. Moreover, two signaling molecules, AKT and p38 MAPK, were activated during the course of apoptosis induction. Employing MTT analysis, we found that a p38 MAPK inhibitor but not an AKT inhibitor could rescue cells from CM-mediated cell death, as well as inhibit the cleavage of PARP, formation of apoptotic bodies and up-regulation of the γH2AX signal. These results suggest that CM-mediated leukemia cell death occurs through the activation of the p38 MAPK pathway, indicating its potential therapeutic effects against human leukemia. PMID:26205966

  20. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation

    PubMed Central

    García-Álvaro, María; Addante, Annalisa; Roncero, Cesáreo; Fernández, Margarita; Fabregat, Isabel; Sánchez, Aránzazu; Herrera, Blanca

    2015-01-01

    The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells. PMID:26343646

  1. Cadmium induces vascular permeability via activation of the p38 MAPK pathway

    SciTech Connect

    Dong, Fengyun; Guo, Fang; Li, Liqun; Guo, Ling; Hou, Yinglong; Hao, Enkui; Yan, Suhua; Allen, Thaddeus D.; Liu, Ju

    2014-07-18

    Highlights: • Low-dose cadmium (Cd) induces vascular hyper-permeability. • p38 MAPK mediates Cd-induced disruption of endothelial cell barrier function. • SB203850 inhibits Cd-induced membrane dissociation of VE-cadherin and β-catenin. • SB203850 reduces Cd-induced expression and secretion of TNF-α. - Abstract: The vasculature of various organs is a targeted by the environmental toxin, cadmium (Cd). However, mechanisms leading to pathological conditions are poorly understood. In the present study, we examined the effect of cadmium chloride (CdCl{sub 2}) on human umbilical vein endothelial cells (HUVECs). At 4 μM, CdCl{sub 2} induced a hyper-permeability defect in HUVECs, but not the inhibition of cell growth up to 24 h. This effect of CdCl{sub 2} was dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The p38 MAPK inhibitor SB203850 suppressed the CdCl{sub 2}-induced alteration in trans-endothelial electrical resistance in HUVEC monolayers, a model measurement of vascular endothelial barrier integrity. SB203850 also inhibited the Cd-induced membrane dissociation of vascular endothelial (VE) cadherin and β-catenin, the important components of the adherens junctional complex. In addition, SB203850 reduces the Cd-induced expression and secretion of tumor necrosis factor α (TNF-α). Taken together, our findings suggest that Cd induces vascular hyper-permeability and disruption of endothelial barrier integrity through stimulation of p38 MAPK signaling.

  2. Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells.

    PubMed

    Leiba, M; Cahalon, L; Shimoni, A; Lider, O; Zanin-Zhorov, A; Hecht, I; Sela, U; Vlodavsky, I; Nagler, A

    2006-08-01

    Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone, P = 0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent. PMID:16769768

  3. BMP7 retards peripheral myelination by activating p38 MAPK in Schwann cells

    PubMed Central

    Liu, Xiaoyu; Zhao, Yahong; Peng, Su; Zhang, Shuqiang; Wang, Meihong; Chen, Yeyue; Zhang, Shan; Yang, Yumin; Sun, Cheng

    2016-01-01

    Schwann cell (SC) myelination is pivotal for the proper physiological functioning of the nervous system, but the underlying molecular mechanism remains less well understood. Here, we showed that the expression of bone morphogenetic protein 7 (BMP7) inversely correlates with myelin gene expression during peripheral myelination, which suggests that BMP7 is likely a negative regulator for myelin gene expression. Our experiments further showed that the application of BMP7 attenuates the cAMP induced myelin gene expression in SCs. Downstream pathway analysis suggested that both p38 MAPK and SMAD are activated by exogenous BMP7 in SCs. The pharmacological intervention and gene silence studies revealed that p38 MAPK, not SMAD, is responsible for BMP7-mediated suppression of myelin gene expression. In addition, c-Jun, a potential negative regulator for peripheral myelination, was up-regulated by BMP7. In vivo experiments showed that BMP7 treatment greatly impaired peripheral myelination in newborn rats. Together, our results established that BMP7 is a negative regulator for peripheral myelin gene expression and that p38 MAPK/c-Jun axis might be the main downstream target of BMP7 in this process. PMID:27491681

  4. BMP7 retards peripheral myelination by activating p38 MAPK in Schwann cells.

    PubMed

    Liu, Xiaoyu; Zhao, Yahong; Peng, Su; Zhang, Shuqiang; Wang, Meihong; Chen, Yeyue; Zhang, Shan; Yang, Yumin; Sun, Cheng

    2016-08-05

    Schwann cell (SC) myelination is pivotal for the proper physiological functioning of the nervous system, but the underlying molecular mechanism remains less well understood. Here, we showed that the expression of bone morphogenetic protein 7 (BMP7) inversely correlates with myelin gene expression during peripheral myelination, which suggests that BMP7 is likely a negative regulator for myelin gene expression. Our experiments further showed that the application of BMP7 attenuates the cAMP induced myelin gene expression in SCs. Downstream pathway analysis suggested that both p38 MAPK and SMAD are activated by exogenous BMP7 in SCs. The pharmacological intervention and gene silence studies revealed that p38 MAPK, not SMAD, is responsible for BMP7-mediated suppression of myelin gene expression. In addition, c-Jun, a potential negative regulator for peripheral myelination, was up-regulated by BMP7. In vivo experiments showed that BMP7 treatment greatly impaired peripheral myelination in newborn rats. Together, our results established that BMP7 is a negative regulator for peripheral myelin gene expression and that p38 MAPK/c-Jun axis might be the main downstream target of BMP7 in this process.

  5. OsMAPK6, a mitogen-activated protein kinase, influences rice grain size and biomass production.

    PubMed

    Liu, Shuying; Hua, Lei; Dong, Sujun; Chen, Hongqi; Zhu, Xudong; Jiang, Jun'e; Zhang, Fang; Li, Yunhai; Fang, Xiaohua; Chen, Fan

    2015-11-01

    Grain size is an important agronomic trait in determining grain yield. However, the molecular mechanisms that determine the final grain size are not well understood. Here, we report the functional analysis of a rice (Oryza sativa L.) mutant, dwarf and small grain1 (dsg1), which displays pleiotropic phenotypes, including small grains, dwarfism and erect leaves. Cytological observations revealed that the small grain and dwarfism of dsg1 were mainly caused by the inhibition of cell proliferation. Map-based cloning revealed that DSG1 encoded a mitogen-activated protein kinase (MAPK), OsMAPK6. OsMAPK6 was mainly located in the nucleus and cytoplasm, and was ubiquitously distributed in various organs, predominately in spikelets and spikelet hulls, consistent with its role in grain size and biomass production. As a functional kinase, OsMAPK6 interacts strongly with OsMKK4, indicating that OsMKK4 is likely to be the upstream MAPK kinase of OsMAPK6 in rice. In addition, hormone sensitivity tests indicated that the dsg1 mutant was less sensitive to brassinosteroids (BRs). The endogenous BR levels were reduced in dsg1, and the expression of several BR signaling pathway genes and feedback-inhibited genes was altered in the dsg1 mutant, with or without exogenous BRs, indicating that OsMAPK6 may contribute to influence BR homeostasis and signaling. Thus, OsMAPK6, a MAPK, plays a pivotal role in grain size in rice, via cell proliferation, and BR signaling and homeostasis. PMID:26366992

  6. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    PubMed Central

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-01-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes. PMID:26096612

  7. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    NASA Astrophysics Data System (ADS)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  8. Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival.

    PubMed

    Wu, Wei; Pew, Travis; Zou, Min; Pang, Diana; Conzen, Suzanne D

    2005-02-11

    Glucocorticoid receptor (GR) activation has recently been shown to inhibit apoptosis in breast epithelial cells. We have previously described a group of genes that is rapidly up-regulated in these cells following dexamethasone (Dex) treatment. In an effort to dissect the mechanisms of GR-mediated breast epithelial cell survival, we now examine the molecular events downstream of GR activation. Here we show that GR activation leads to both the rapid induction of MAPK phosphatase-1 (MKP-1) mRNA and its sustained expression. Induction of the MKP-1 protein in the MCF10A-Myc and MDA-MB-231 breast epithelial cell lines was also seen. Paclitaxel treatment resulted in MAPK activation and apoptosis of MDA-MB-231 breast cancer cells, and both processes were inhibited by Dex pretreatment. Furthermore, induction of MKP-1 correlated with the inhibition of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) activity, whereas p38 activity was minimally affected. Blocking Dex-induced MKP-1 induction using small interfering RNA increased ERK1/2 and JNK phosphorylation and decreased cell survival. ERK1/2 and JNK inactivation was associated with Ets-like transcription factor-1 (ELK-1) dephosphorylation. To explore the gene expression changes that occur downstream of ELK-1 dephosphorylation, we used a combination of temporal gene expression data and promoter element analyses. This approach revealed a previously unrecognized transcriptional target of ELK-1, the human tissue plasminogen activator (tPA). We verified the predicted ELK-1--> tPA transcriptional regulatory relationship using a luciferase reporter assay. We conclude that GR-mediated MAPK inactivation contributes to cell survival and that the potential transcriptional targets of this inhibition can be identified from large scale gene array analysis.

  9. Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

    PubMed

    Centuori, Sara M; Gomes, Cecil J; Trujillo, Jesse; Borg, Jamie; Brownlee, Joshua; Putnam, Charles W; Martinez, Jesse D

    2016-07-01

    Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.

  10. The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells

    PubMed Central

    Park, Seon-Joo; Kim, In-Sook

    2005-01-01

    In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 μM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells. PMID:16086031

  11. Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

    PubMed

    Centuori, Sara M; Gomes, Cecil J; Trujillo, Jesse; Borg, Jamie; Brownlee, Joshua; Putnam, Charles W; Martinez, Jesse D

    2016-07-01

    Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects. PMID:27086143

  12. Increase of mesenchymal stem cell migration by cannabidiol via activation of p42/44 MAPK.

    PubMed

    Schmuhl, Ellen; Ramer, Robert; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2014-02-01

    Migration and differentiation of mesenchymal stem cells (MSCs) are known to be involved in various regenerative processes such as bone healing. However, little is known about the pharmacotherapeutical options aiming at the mobilization and differentiation of MSCs. The present study therefore focussed on cannabinoids which have been demonstrated to exhibit tissue healing properties. Using Boyden chamber assays, the non-psychoactive phytocannabinoid cannabidiol (CBD) was found to increase the migration of adipose-derived MSCs in a time- and concentration-dependent manner. CBD-induced migration was inhibited by AM-630 (CB₂ receptor antagonist) and O-1602 (G protein-coupled receptor 55 [GRP55] agonist). Moreover, the promigratory effect of CBD was antagonized by inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway which became activated upon CBD treatment. In line with this data, AM-630 and O-1602 attenuated CBD-induced p42/44 MAPK phosphorylation. A p42/44 MAPK-dependent promigratory effect was likewise demonstrated for the GPR55 antagonist O-1918 and the selective CB₂ receptor agonist JWH-133. Additional evidence for a functional effect of CBD on MSCs was provided by experiments demonstrating long-term stimulation with CBD to induce differentiation of MSCs into the osteoblastic lineage as evidenced by increased mineralization assessed by cresolphthalein complexone assay and enhanced activity of alkaline phosphatase. Collectively, this study demonstrates CBD to promote the migration of MSCs via activation of the CB₂ receptor and inhibition of GPR55 and to induce osteoblastic differentiation. CBD may therefore recruit MSCs to sites of calcifying tissue regeneration and subsequently support bone regeneration via an osteoanabolic action on MSCs.

  13. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

    PubMed Central

    Medina-Castellanos, Elizabeth; Esquivel-Naranjo, Edgardo U.; Heil, Martin; Herrera-Estrella, Alfredo

    2014-01-01

    The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals. PMID:25484887

  14. Aluminum Nanoparticles Induce ERK and p38MAPK Activation in Rat Brain

    PubMed Central

    Seo, Gyun-Baek; Jo; Lee, Mimi; Shim, Ilseob; Lee, Byung-Woo; Yoon, Byung-Il; Kim, Pilje; Choi, Kyunghee

    2013-01-01

    Aluminum nanoparticles (Al-NPs) are one of the most widely used nanomaterial in cosmetics and medical materials. For this reason, Al-NP exposure is very likely to occur via inhalation in the environment and the workplace. Nevertheless, little is known about the mechanism of Al-NP neurotoxicity via inhalation exposure. In this study, we investigated the effect AL-NPs on the brain. Rats were exposed to Al-NPs by nasal instillation at 1 mg/kg body weight (low exposure group), 20 mg/kg body weight (moderate exposure group), and 40 mg/kg body weight (high exposure group), for a total of 3 times, with a 24-hr interval after each exposure. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated that the presence of aluminum was increased in a dose-dependent manner in the olfactory bulb (OFB) and the brain. In microarray analysis, the regulation of mitogen-activated protein kinases (MAPK) activity (GO: 0043405), including Ptprc, P2rx7, Map2k4, Trib3, Trib1, and Fgd4 was significantly over-expressed in the treated mice than in the controls (p = 0.0027). Moreover, Al-NPs induced the activation of ERK1 and p38 MAPK protein expression in the brain, but did not alter the protein expression of JNK, when compared to the control. These data demonstrate that the nasal exposure of Al-NPs can permeate the brain via the olfactory bulb and modulate the gene and protein expression of MAPK and its activity. PMID:24386518

  15. Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics

    PubMed Central

    Li, Ling-bo; Leung, Donald Y. M.; Goleva, Elena

    2015-01-01

    Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14+ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14+ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid

  16. PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction.

    PubMed

    Gonnella, Roberta; Granato, Marisa; Farina, Antonella; Santarelli, Roberta; Faggioni, Alberto; Cirone, Mara

    2015-07-01

    PKC activation by combining TPA with sodium butyrate (T/B) represents the most effective and widely used strategy to induce the Epstein-Barr virus (EBV) lytic cycle. The results obtained in this study show that novel PKCθ is involved in such process and that it acts through the activation of p38 MAPK and autophagy induction. Autophagy, a mechanism of cellular defense in stressful conditions, is manipulated by EBV to enhance viral replication. Besides promoting the EBV lytic cycle, the activation of p38 and autophagy resulted in a pro-survival effect, as indicated by p38 or ATG5 knocking down experiments. However, this pro-survival role was counteracted by a pro-death activity of PKCθ, due to the dephosphorylation of AKT. In conclusion, this study reports, for the first time, that T/B activates a PKCθ-p38 MAPK axis in EBV infected B cells, that promotes the viral lytic cycle and cell survival and dephosphorylates AKT, balancing cell life and cell death. PMID:25827954

  17. Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

    PubMed Central

    Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong

    2014-01-01

    Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders. PMID:24868545

  18. Hydrogen peroxide signaling mediator in the activation of p38 MAPK in vascular endothelial cells.

    PubMed

    Bretón-Romero, Rosa; Lamas, Santiago

    2013-01-01

    Substantial evidence suggests that a transient increase of hydrogen peroxide (H2O2) behaves as an intracellular messenger able to trigger the activation of different signaling pathways. These include phosphatases, protein kinases, and transcription factors among others; however, most of the studies have been performed using supraphysiological levels of H2O2. Reactive oxygen species (ROS) generation occurs under physiological conditions and different extracellular stimuli including cytokines, growth factors, and shear stress are able to produce both low levels of superoxide anion and H2O2. Here, we explore the redox-dependent activation of key signaling pathways induced by shear stress. We demonstrate that laminar shear stress (LSS) rapidly promotes a transient generation of H2O2 that is necessary for the activation of the stress-activated protein kinase p38 MAPK. We describe p38 MAPK as an early redox sensor in LSS. Our studies show that it is essential for the activation of endothelial nitric oxide synthase, the subsequent nitric oxide generation, and the protection of endothelial function.

  19. PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction.

    PubMed

    Gonnella, Roberta; Granato, Marisa; Farina, Antonella; Santarelli, Roberta; Faggioni, Alberto; Cirone, Mara

    2015-07-01

    PKC activation by combining TPA with sodium butyrate (T/B) represents the most effective and widely used strategy to induce the Epstein-Barr virus (EBV) lytic cycle. The results obtained in this study show that novel PKCθ is involved in such process and that it acts through the activation of p38 MAPK and autophagy induction. Autophagy, a mechanism of cellular defense in stressful conditions, is manipulated by EBV to enhance viral replication. Besides promoting the EBV lytic cycle, the activation of p38 and autophagy resulted in a pro-survival effect, as indicated by p38 or ATG5 knocking down experiments. However, this pro-survival role was counteracted by a pro-death activity of PKCθ, due to the dephosphorylation of AKT. In conclusion, this study reports, for the first time, that T/B activates a PKCθ-p38 MAPK axis in EBV infected B cells, that promotes the viral lytic cycle and cell survival and dephosphorylates AKT, balancing cell life and cell death.

  20. TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function

    PubMed Central

    Lee, Guan-Lin; Wu, Jing-Yiing; Tsai, Chien-Sung; Lin, Chih-Yuan; Tsai, Yi-Ting; Lin, Chin-Sheng; Wang, Yi-Fu; Yet, Shaw-Fang; Hsu, Yu-Juei; Kuo, Cheng-Chin

    2016-01-01

    Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration. PMID:27563891

  1. TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function.

    PubMed

    Lee, Guan-Lin; Wu, Jing-Yiing; Tsai, Chien-Sung; Lin, Chih-Yuan; Tsai, Yi-Ting; Lin, Chin-Sheng; Wang, Yi-Fu; Yet, Shaw-Fang; Hsu, Yu-Juei; Kuo, Cheng-Chin

    2016-01-01

    Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration. PMID:27563891

  2. Nobiletin suppresses MMP-9 expression through modulation of p38 MAPK activity in human dermal fibrobalsts.

    PubMed

    Kim, Jin-Ju; Korm, Sovannarith; Kim, Won-Seok; Kim, Ok-Seon; Lee, Ji-Seon; Min, Hyung-Geun; Chin, Young-Won; Cha, Hyuk-Jin

    2014-01-01

    We aimed to identify a novel flavonoid from the in-house natural products to suppress matrix metalloproteases (MMPs), which is responsible for degradation of collagen and other extracellular matrix proteins. Total eight natural products were screened for identification of a novel MMP-9 suppressor using MMP-9 reporter system, where the prompt initial screening with multiple samples is readily examined. Among the extracts used in the present study, one extract (Citrus unshiu) was found active in this assay system. Furthermore, three representative flavonoids in this active extract of Citrus unshiu peel were tested in MMP-9 reporter system. Nobiletin (NB) of the tested flavonoids suppressed MMP-9 expression without cytotoxicity, which was validated by both real-time polymerase chain reaction (PCR) and zymography analyses. Sustained p38 mitogen activated protein kinase (MAPK) activity, closely associated with induction of MMP-9 under stress condition, was markedly reduced by NB treatment, which implies that modulation of p38MAPK by nobiletin is responsible for reduction of MMP9 expression. Hence, nobiletin, identified from MMP-9 reporter system based screening, may be further applied for the purpose of delaying collagen degradation in skin fibroblasts.

  3. MEK Inhibition Overcomes Cisplatin Resistance Conferred by SOS/MAPK Pathway Activation in Squamous Cell Carcinoma.

    PubMed

    Kong, Li Ren; Chua, Kian Ngiap; Sim, Wen Jing; Ng, Hsien Chun; Bi, Chonglei; Ho, Jingshan; Nga, Min En; Pang, Yin Huei; Ong, Weijie Richard; Soo, Ross Andrew; Huynh, Hung; Chng, Wee Joo; Thiery, Jean-Paul; Goh, Boon Cher

    2015-07-01

    Genomic analyses of squamous cell carcinoma (SCC) have yet to yield significant strategies against pathway activation to improve treatment. Platinum-based chemotherapy remains the mainstay of treatment for SCC of different histotypes either as a single-agent or alongside other chemotherapeutic drugs or radiotherapy; however, resistance inevitably emerges, which limits the duration of treatment response. To elucidate mechanisms that mediate resistance to cisplatin, we compared drug-induced perturbations to gene and protein expression between cisplatin-sensitive and -resistant SCC cells, and identified MAPK-ERK pathway upregulation and activation in drug-resistant cells. ERK-induced resistance appeared to be activated by Son of Sevenless (SOS) upstream, and mediated through Bim degradation downstream. Clinically, elevated p-ERK expression was associated with shorter disease-free survival in patients with locally advanced head and neck SCC treated with concurrent chemoradiation. Inhibition of MEK/ERK, but not that of EGFR or RAF, augmented cisplatin sensitivity in vitro and demonstrated efficacy and tolerability in vivo. Collectively, these findings suggest that inhibition of the activated SOS-MAPK-ERK pathway may augment patient responses to cisplatin treatment.

  4. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis.

    PubMed

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J; Lipschutz, Joshua H

    2014-12-15

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.

  5. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis

    PubMed Central

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M.; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F.; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J.

    2014-01-01

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury. PMID:25298525

  6. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa

    PubMed Central

    Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

  7. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa.

    PubMed

    Lu, Kun; Guo, Wenjin; Lu, Junxing; Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa.

  8. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa.

    PubMed

    Lu, Kun; Guo, Wenjin; Lu, Junxing; Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

  9. Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation

    PubMed Central

    Chu, Ling-Yun; Wang, Yi-Fu; Cheng, Huei-Hsuan; Kuo, Cheng-Chin; Wu, Kenneth K.

    2016-01-01

    The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK. PMID:27002329

  10. Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation.

    PubMed

    Chu, Ling-Yun; Wang, Yi-Fu; Cheng, Huei-Hsuan; Kuo, Cheng-Chin; Wu, Kenneth K

    2016-01-01

    The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.

  11. Transforming growth factor-beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.

    PubMed

    Reimann, T; Hempel, U; Krautwald, S; Axmann, A; Scheibe, R; Seidel, D; Wenzel, K W

    1997-02-10

    The transdifferentiation of hepatic stellate cells into myofibroblast-like cells and the proliferation of the transdifferentiated cells are controlled by TGF-beta1. Little is known about the intracellular signal transducers of TGF-beta1. In this paper we show that in cultured hepatic stellate cells TGF-beta1 induces activation of Ras, Raf-1, MEK and MAPK p42 and p44. The activation of MAPK depends on the activation of MEK. Our data exclude that the observed effects are mediated by a bFGF or PDGF autocrine loop. PMID:9038360

  12. Homology modeling studies of yeast Mitogen-Activated Protein Kinases (MAPKS): structural motifs as a basis for specificity.

    PubMed

    Smith, D L; Nilar, S H

    2010-06-01

    Mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction. It is the objective of this communication to demonstrate that insight into protein-protein interactions in the Common Docking motif of yeast mitogen-activated protein kinases can be obtained based on homology models. Homology models for four yeast MAPKs, FUS3, KSS1, HOG1 and MPK1 were built based on the X-ray structures of active and inactive rat ERK2. The structural motifs required for the basis of specificity were rationalized based on these structures. PMID:19995338

  13. IGF-1 protects tubular epithelial cells during injury via activation of ERK/MAPK signaling pathway

    PubMed Central

    Wu, Zengbin; Yu, Yang; Niu, Lei; Fei, Aihua; Pan, Shuming

    2016-01-01

    Injury of renal tubular epithelial cells can induce acute renal failure and obstructive nephropathy. Previous studies have shown that administration of insulin-like growth factor-1 (IGF-1) ameliorates the renal injury in a mouse unilateral ureteral obstruction (UUO) model, whereas the underlying mechanisms are not completely understood. Here, we addressed this question. We found that the administration of IGF-1 significantly reduced the severity of the renal fibrosis in UUO. By analyzing purified renal epithelial cells, we found that IGF-1 significantly reduced the apoptotic cell death of renal epithelial cells, seemingly through upregulation of anti-apoptotic protein Bcl-2, at protein but not mRNA level. Bioinformatics analyses and luciferase-reporter assay showed that miR-429 targeted the 3′-UTR of Bcl-2 mRNA to inhibit its protein translation in renal epithelial cells. Moreover, IGF-1 suppressed miR-429 to increase Bcl-2 in renal epithelial cells to improve survival after UUO. Furthermore, inhibition of ERK/MAPK signaling pathway in renal epithelial cells abolished the suppressive effects of IGF-1 on miR-429 activation, and then the enhanced effects on Bcl-2 in UUO. Thus, our data suggest that IGF-1 may protect renal tubular epithelial cells via activation of ERK/MAPK signaling pathway during renal injury. PMID:27301852

  14. Differential Activation of Mitogen-Activated Protein Kinases, ERK 1/2, p38(MAPK) and JNK p54/p46 During Postnatal Development of Rat Hippocampus.

    PubMed

    Costa, Ana Paula; Lopes, Mark William; Rieger, Débora K; Barbosa, Sabrina Giovana Rocha; Gonçalves, Filipe Marques; Xikota, João Carlos; Walz, Roger; Leal, Rodrigo B

    2016-05-01

    Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38(MAPK), ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38(MAPK) at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38(MAPK) during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38(MAPK) are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus.

  15. Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling.

    PubMed

    Shi, Yu; Xia, Yin-Yan; Wang, Lei; Liu, Rui; Khoo, King-Shung; Feng, Zhi-Wei

    2012-10-15

    Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.

  16. MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

    SciTech Connect

    Liu, Lian; Zhang, Xin; Du, Chao; Zhang, Xiaoning; Hou, Nan; Zhao, Di; Sun, Jianzhi; Li, Li; Wang, Xiuwen; Ma, Chunhong

    2009-05-01

    We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even when it fails to activate MAPK as expected.

  17. Interferon-β counter-regulates its own pro-apoptotic action by activating p38 MAPK signalling in human SH-SY5Y neuroblastoma cells.

    PubMed

    Dedoni, Simona; Olianas, Maria C; Onali, Pierluigi

    2014-10-01

    Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-β induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-β-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-β-induced apoptosis of neuroblastoma cells. Exposure to IFN-β increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-β-induced apoptotic death. These results indicate that IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.

  18. RAS/MAPK activation drives resistance to Smo inhibition, metastasis and tumor evolution in Shh pathway-dependent tumors

    PubMed Central

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J.; Zhou, Pengcheng; Dabral, Sukriti K.; Pak, Ekaterina; Li, Wei; Atwood, Scott X.; Whitson, Ramon J.; Chang, Anne Lynn S.; Li, Jiang; Oro, Anthony E.; Chan, Jennifer A.; Kelleher, Joseph F.; Segal, Rosalind A.

    2015-01-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS/MAPK pathway circumvents Shh pathway-dependency, drives tumor growth and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together these findings reveal a critical role of RAS/MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  19. Michael addition of dehydroalanine-containing MAPK peptides to catalytic lysine inhibits the activity of phosphothreonine lyase.

    PubMed

    Zhang, Yuan; Yang, Ru; Huang, Juan; Liang, Qiujin; Guo, Yanmin; Bian, Weixiang; Luo, Lingfei; Li, Hongtao

    2015-11-30

    The phosphothreonine lyases OspF and SpvC irreversibly inactivate host dual-phosphorylated mitogen-activated protein kinases (MAPKs) [pThr-X-pTyr motif] through β-elimination. We found that dual-phosphorylated (pSer-X-pTyr) MAPK substrate peptides and their resulting catalytic products cross-link to OspF and SpvC. Mass spectrometry results revealed that these linkages form between lysine, which acts as a general base, and dehydroalanine (Dha) on catalytic products. The nucleophilic addition efficiency is dependent on the K136 residue being in a deprotonated state. Peptide cross-linking inhibits the activity of SpvC and blocks the inactivation of MAPK signaling by SpvC. Small compounds mimicking these sequences may act as phosphothreonine lyase inhibitors. PMID:26519561

  20. N-methyl-D-aspartate (NMDA) and the regulation of mitogen-activated protein kinase (MAPK) signaling pathways: a revolving neurochemical axis for therapeutic intervention?

    PubMed

    Haddad, John J

    2005-11-01

    Excitatory synaptic transmission in the central nervous system (CNS) is mediated by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and non-NMDA (AMPA and KA) receptors. Extracellular signals control diverse neuronal functions and are responsible for mediating activity-dependent changes in synaptic strength and neuronal survival. Influx of extracellular calcium ([Ca(2+)](e)) through the NMDA receptor (NMDAR) is required for neuronal activity to change the strength of many synapses. At the molecular level, the NMDAR interacts with signaling modules, which, like the mitogen-activated protein kinase (MAPK) superfamily, transduce excitatory signals across neurons. Recent burgeoning evidence points to the fact that MAPKs play a crucial role in regulating the neurochemistry of NMDARs, their physiologic and biochemical/biophysical properties, and their potential role in pathophysiology. It is the purpose of this review to discuss: (i) the MAPKs and their role in a plethora of cellular functions; (ii) the role of MAPKs in regulating the biochemistry and physiology of NMDA receptors; (iii) the kinetics of MAPK-NMDA interactions and their biologic and neurochemical properties; (iv) how cellular signaling pathways, related cofactors and intracellular conditions affect NMDA-MAPK interactions and (v) the role of NMDA-MAPK pathways in pathophysiology and the evolution of disease conditions. Given the versatility of the NMDA-MAPK interactions, the NMDA-MAPK axis will likely form a neurochemical target for therapeutic interventions.

  1. Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

    PubMed

    Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Darlyuk-Saadon, Ilona; Bai, Chen; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-09-01

    MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect. PMID:27413009

  2. Small Molecule APY606 Displays Extensive Antitumor Activity in Pancreatic Cancer via Impairing Ras-MAPK Signaling

    PubMed Central

    Guo, Na; Liu, Zuojia; Zhao, Wenjing; Wang, Erkang; Wang, Jin

    2016-01-01

    Pancreatic cancer has been found with abnormal expression or mutation in Ras proteins. Oncogenic Ras activation exploits their extensive signaling reach to affect multiple cellular processes, in which the mitogen-activated protein kinase (MAPK) signaling exerts important roles in tumorigenesis. Therapies targeted Ras are thus of major benefit for pancreatic cancer. Although small molecule APY606 has been successfully picked out by virtual drug screening based on Ras target receptor, its in-depth mechanism remains to be elucidated. We herein assessed the antitumor activity of APY606 against human pancreatic cancer Capan-1 and SW1990 cell lines and explored the effect of Ras-MAPK and apoptosis-related signaling pathway on the activity of APY606. APY606 treatment resulted in a dose- and time-dependent inhibition of cancer cell viability. Additionally, APY606 exhibited strong antitumor activity, as evidenced not only by reduction in tumor cell invasion, migration and mitochondrial membrane potential but also by alteration in several apoptotic indexes. Furthermore, APY606 treatment directly inhibited Ras-GTP and the downstream activation of MAPK, which resulted in the down-regulation of anti-apoptotic protein Bcl-2, leading to the up-regulation of mitochondrial apoptosis pathway-related proteins (Bax, cytosolic Cytochrome c and Caspase 3) and of cyclin-dependent kinase 2 and Cyclin A, E. These data suggest that impairing Ras-MAPK signaling is a novel mechanism of action for APY606 during therapeutic intervention in pancreatic cancer. PMID:27223122

  3. Trientine, a copper-chelating agent, induced apoptosis in murine fibrosarcoma cells by activation of the p38 MAPK pathway.

    PubMed

    KADOWAKI, Shingo; ENDOH, Daiji; OKUI, Toyo; HAYASHI, Masanobu

    2009-11-01

    We have reported that treatment with trientine, Cu-chelating agent, inhibits tumor growth in a murine transplantation model using fibrosarcoma and induces apoptosis in tumor cells in vivo and in vitro. When fibrosarcoma cells were treated with 10 mM trientine, the activities of p38 MAPK in treated cells were approximately 3-4 times higher than those in untreated cells. Proportions of cells in which apoptosis was induced by trientine increased in an incubation time-dependent manner from days 2 to 6. The proportions of apoptotic cells in the cells treated with trientine and SB203580, an inhibitor of p38 MAPK, were approximately 50% in those of cells treated with trientine alone. The present results showed that the p38 MAPK pathway may play an important role in induction of apoptosis in fibrosarcoma cells by trientine.

  4. Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity

    PubMed Central

    Mota, Caroline M.; Oliveira, Ana C. M.; Davoli-Ferreira, Marcela; Silva, Murilo V.; Santiago, Fernanda M.; Nadipuram, Santhosh M.; Vashisht, Ajay A.; Wohlschlegel, James A.; Bradley, Peter J.; Silva, João S.; Mineo, José R.; Mineo, Tiago W. P.

    2016-01-01

    Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii’s GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host’s innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis. PMID:27679624

  5. Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity

    PubMed Central

    Mota, Caroline M.; Oliveira, Ana C. M.; Davoli-Ferreira, Marcela; Silva, Murilo V.; Santiago, Fernanda M.; Nadipuram, Santhosh M.; Vashisht, Ajay A.; Wohlschlegel, James A.; Bradley, Peter J.; Silva, João S.; Mineo, José R.; Mineo, Tiago W. P.

    2016-01-01

    Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii’s GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host’s innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.

  6. Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

    PubMed

    Mota, Caroline M; Oliveira, Ana C M; Davoli-Ferreira, Marcela; Silva, Murilo V; Santiago, Fernanda M; Nadipuram, Santhosh M; Vashisht, Ajay A; Wohlschlegel, James A; Bradley, Peter J; Silva, João S; Mineo, José R; Mineo, Tiago W P

    2016-01-01

    Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis. PMID:27679624

  7. Hypoxia differentially regulates the mitogen- and stress-activated protein kinases. Role of Ca2+/CaM in the activation of MAPK and p38 gamma.

    PubMed

    Conrad, P W; Millhorn, D E; Beitner-Johnson, D

    2000-01-01

    Hypoxic/ischemic trauma is a primary factor in the pathology of various vascular, pulmonary, and cerebral disease states. Yet, the signaling mechanisms by which cells respond and adapt to changes in oxygen levels are not clearly established. The effects of hypoxia on the stress- and mitogen-activated protein kinase (SAPK and MAPK) signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O2) was found to progressively stimulate phosphorylation and activation of p38 gamma in particular, and also p38 alpha, two isoforms of the p38 family of stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38 beta, p38 beta 2, p38 delta, or on JNK, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 MAPK, although this activation was modest when compared to NGF and UV-induced activation. We further showed that activation of p38 gamma and MAPK during hypoxia requires calcium, as treatment with Ca(2+)-free media or the calmodulin antagonist, W13, blocked the activation of p38 gamma and MAPK, respectively. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific elements of the SAPKs and MAPKs, and identifies Ca+2/CaM as a critical upstream activator. PMID:10849670

  8. CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

    PubMed

    Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

    2016-08-26

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease.

  9. CONTRIBUTION OF INSPIRATORY FLOW TO ACTIVATION OF EGFR, RAS, MAPK, ATF-2 AND C-JUN DURING LUNG STRETCH

    EPA Science Inventory

    Contribution of Inspiratory Flow to Activation of EGFR, Ras, MAPK, ATF-2 and c-Jun during Lung Stretch

    R. Silbajoris 1, Z. Li 2, J. M. Samet 1 and Y. C. Huang 1. 1 NHEERL, ORD, US EPA, RTP, NC and 2 CEMALB, UNC-CH, Chapel Hill, NC .

    Mechanical ventilation with larg...

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

    PubMed Central

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

    2013-01-01

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

  11. Increases in cAMP, MAPK activity, and CREB phosphorylation during REM sleep: implications for REM sleep and memory consolidation.

    PubMed

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

    2013-04-10

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

  12. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  13. Importin-7 mediates memory consolidation through regulation of nuclear translocation of training-activated MAPK in Drosophila.

    PubMed

    Li, Qian; Zhang, Xuchen; Hu, Wantong; Liang, Xitong; Zhang, Fang; Wang, Lianzhang; Liu, Zhong-Jian; Zhong, Yi

    2016-03-15

    Translocation of signaling molecules, MAPK in particular, from the cytosol to nucleus represents a universal key element in initiating the gene program that determines memory consolidation. Translocation mechanisms and their behavioral impact, however, remain to be determined. Here, we report that a highly conserved nuclear transporter, Drosophila importin-7 (DIM-7), regulates import of training-activated MAPK for consolidation of long-term memory (LTM). We show that silencing DIM-7 functions results in impaired LTM, whereas overexpression of DIM-7 enhances LTM. This DIM-7-dependent regulation of LTM is confined to a consolidation time window and in mushroom body neurons. Image data show that bidirectional alteration in DIM-7 expression results in proportional changes in the intensity of training-activated MAPK accumulated within the nuclei of mushroom body neurons during LTM consolidation. Such DIM-7-regulated nuclear accumulation of activated MAPK is observed only in the training specified for LTM induction and determines the amplitude, but not the time course, of memory consolidation. PMID:26929354

  14. Importin-7 mediates memory consolidation through regulation of nuclear translocation of training-activated MAPK in Drosophila

    PubMed Central

    Li, Qian; Zhang, Xuchen; Liang, Xitong; Zhang, Fang; Wang, Lianzhang; Zhong, Yi

    2016-01-01

    Translocation of signaling molecules, MAPK in particular, from the cytosol to nucleus represents a universal key element in initiating the gene program that determines memory consolidation. Translocation mechanisms and their behavioral impact, however, remain to be determined. Here, we report that a highly conserved nuclear transporter, Drosophila importin-7 (DIM-7), regulates import of training-activated MAPK for consolidation of long-term memory (LTM). We show that silencing DIM-7 functions results in impaired LTM, whereas overexpression of DIM-7 enhances LTM. This DIM-7–dependent regulation of LTM is confined to a consolidation time window and in mushroom body neurons. Image data show that bidirectional alteration in DIM-7 expression results in proportional changes in the intensity of training-activated MAPK accumulated within the nuclei of mushroom body neurons during LTM consolidation. Such DIM-7–regulated nuclear accumulation of activated MAPK is observed only in the training specified for LTM induction and determines the amplitude, but not the time course, of memory consolidation. PMID:26929354

  15. Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells.

    PubMed

    Reddy, K B; Krueger, J S; Kondapaka, S B; Diglio, C A

    1999-07-19

    Mitogen-activated protein kinases (MAPKs) play a major role in the mitogenic signal transduction pathway and are essential components of both growth and differentiation. Constitutive activation of the MAPK cascade is associated with the carcinogenesis and metastasis of human breast and renal cell carcinomas. The gelatinases B (MMP-9) and A (MMP-2) are 2 members of the matrix metalloproteinase (MMPs) family which are expressed in human cancers and thought to play a critical role in tumor cell invasion and metastasis. In a previous study, we have shown that EGF and amphiregulin upregulate MMP-9 in metastatic SKBR-3 cells but have no effect on MMP-2 secretion. We now investigated specific step(s) in EGF-induced signalling associated with regulation of cell proliferation and MMP-9 induction. EGF-induced signalling in SKBR-3 cells was blocked by relatively specific inhibitors either on ras (FPT inhibitor-1) or P13 kinase (Wortmannin) or by reduction in EGF-induced tyrosine kinase activity (RG 13022). Blocking these signalling pathways significantly inhibited of EGF-induced cell proliferation but only partially reduced in EGF-induced MMP-9 secretion. In contrast, when SKBR-3 cells were exposed to MEK inhibitor (PD 98059) or MAPK inhibitors (Apigenin or MAPK antisense phosphorothioate oligodeoxynucleotides), EGF-induced cell proliferation, MMP-9 induction and invasion through reconstituted basement membrane were significantly reduced. Our results suggest that interfering with MAPK activity may provide a novel means of controlling growth and invasiveness of tumors in which the signalling cascade is activated.

  16. cAMP blocks MAPK activation and sclerotial development via Rap-1 in a PKA-independent manner in Sclerotinia sclerotiorum.

    PubMed

    Chen, Changbin; Dickman, Martin B

    2005-01-01

    Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen-activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)-independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap-1 or Ras is involved in cAMP action. The Rap-1 inhibitor, GGTI-298, restored MAPK activation in the presence of cAMP, further suggesting that Rap-1 is responsible for cAMP-dependent MAPK inhibition. Importantly, inhibition of Rap-1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA-independent cAMP signalling pathway. Cross-talk between these two pathways is mediated by Rap-1. PMID:15612936

  17. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    PubMed

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  18. Oridonin upregulates PTEN through activating p38 MAPK and inhibits proliferation in human colon cancer cells.

    PubMed

    Wu, Qiu-Xiang; Yuan, Shuang-Xue; Ren, Chun-Mei; Yu, Yu; Sun, Wen-Juan; He, Bai-Cheng; Wu, Ke

    2016-06-01

    Oridonin (ORI) has been reported as an antiproliferation and apoptosis-inducing natural product in various cancer cells. However, the exact molecular mechanism underlying these effects remains unclear. In the present study, we demonstrated the antiproliferation effect of ORI in HCT116 cells, and analyzed the possible molecular mechanism which mediates this effect. We found that ORI inhibits proliferation, induces cell cycle arrest and apoptosis in HCT116 cells, thus also tumor growth. Mechanically, we found that ORI has no substantial effect on mRNA expression of phosphatase and tensin homologue (PTEN), but increases the total protein level of PTEN and markedly reduces the phosphorylation of PTEN; Exogenous expression of PTEN potentiates the anticancer effect of ORI, while knockdown of PTEN attenuates it. ORI also increases the phosphorylation of p38 MAPK, and p38 MAPK-specific inhibitor reduces the antiproliferation effect ORI in HCT116 cells. Moreover, inhibition of p38 MAPK increases the phosphorylation of PTEN, and reverses ORI-induced decrease of PTEN phosphorylation. Our findings suggested that ORI may be a potential anticancer drug for colon cancer, this effect may be mediated by enhancing the function of PTEN through reducing its phosphorylation, which may be resulted from the ORI-induced activation of p38 MAPK.

  19. Hydrogen peroxide mediates hyperglycemia-induced invasive activity via ERK and p38 MAPK in human pancreatic cancer

    PubMed Central

    Ma, Jiguang; Li, Xuqi; Xu, Qinhong; Duan, Wanxing; Chen, Xin; Lv, Yunfu; Zhou, Shuang; Wu, Erxi; Ma, Qingyong; Huo, Xiongwei

    2015-01-01

    Diabetes mellitus and pancreatic cancer are intimately related, as approximately 85% of pancreatic cancer patients suffer from glucose intolerance or even diabetes. In this study, we evaluate the underlying mechanism by which hyperglycemia modulates the invasive potential of cancer cells and contributes to their enhanced metastatic behavior. Here we show that hyperglycemia increases the hydrogen peroxide (H2O2) concentration through up-regulation of manganese superoxide dismutase (SOD2) expression, which further activates the ERK and p38 MAPK pathways, as well as the transcription factors NF-κB and AP-1, in a time-dependent manner. The invasion of pancreatic cancer cells resulting from the activation of the H2O2/MAPK axis under high glucose conditions is effectively inhibited by PD 98059 (ERK inhibitor), SB 203580 (p38 MAPK inhibitor), polyethylene glycol-conjugated catalase (PEG-CAT), or the siRNA specific to SOD2. In addition, streptozotocin-treated diabetic nude mice exhibit a stronger tumor invasive ability in renal capsule xenografts which could be suppressed by PEG-CAT treatment. Furthermore, the integrated optical density (IOD) of SOD2 and uPA stainings is higher in the tumor tissues of pancreatic cancer patients with diabetes compared with pancreatic cancer patients with euglycemia. Taken together, our results demonstrate that hyperglycemia enhances cell invasive ability through the SOD2/H2O2/MAPK axis in human pancreatic cancer. Thus, SOD2/H2O2/MAPK axis may represent a promising therapeutic target for pancreatic cancer patients combined with diabetes mellitus. PMID:26439801

  20. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition.

    PubMed

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D; Nencioni, Alessio

    2015-05-20

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

  1. GNA14 Somatic Mutation Causes Congenital and Sporadic Vascular Tumors by MAPK Activation.

    PubMed

    Lim, Young H; Bacchiocchi, Antonella; Qiu, Jingyao; Straub, Robert; Bruckner, Anna; Bercovitch, Lionel; Narayan, Deepak; McNiff, Jennifer; Ko, Christine; Robinson-Bostom, Leslie; Antaya, Richard; Halaban, Ruth; Choate, Keith A

    2016-08-01

    Vascular tumors are among the most common neoplasms in infants and children; 5%-10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS, KRAS, NRAS, GNAQ, and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention. PMID:27476652

  2. Control of the segmentation process by graded MAPK/ERK activation in the chick embryo.

    PubMed

    Delfini, Marie-Claire; Dubrulle, Julien; Malapert, Pascale; Chal, Jérome; Pourquié, Olivier

    2005-08-01

    The regular spacing of somites during vertebrate embryogenesis involves a dynamic gradient of FGF signaling that controls the timing of maturation of cells in the presomitic mesoderm (PSM). How the FGF signal is transduced by PSM cells is unclear. Here, we first show that the FGF gradient is translated into graded activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway along the PSM in the chicken embryo. Using in ovo electroporation of PSM cells, we demonstrate that constitutive activation of ERK signaling in the PSM blocks segmentation by preventing maturation of PSM cells, thus phenocopying the overexpression of FGF8. Conversely, inhibition of ERK phosphorylation mimics a loss of function of FGF signaling in the PSM. Interestingly, video microscopy analysis of cell movements shows that ERK regulates the motility of PSM cells, suggesting that the decrease of cell movements along the PSM enables mesenchymal PSM cells to undergo proper segmentation. Together, our data demonstrate that ERK is the effector of the gradient of FGF in the PSM that controls the segmentation process.

  3. Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy.

    PubMed

    Slone, Samuel; Anthony, Sarah R; Wu, Xiaoqing; Benoit, Joshua B; Aube, Jeffrey; Xu, Liang; Tranter, Michael

    2016-11-01

    The RNA binding protein Human antigen R (HuR) interacts with specific AU-rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. However, the role of HuR in cardiac physiology is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic cardiac myocytes. Specifically, HuR cytoplasmic translocation is significantly increased in NRVMs (neonatal rat ventricular myocytes) following treatment with phenylephrine or angiotensin II, agonists of two independent Gαq-coupled GPCRs known to induce hypertrophy. This Gq-mediated HuR activation is dependent on p38 MAP kinase, but not canonical Gq-PKC signaling. Furthermore, we show that HuR activation is necessary for Gq-mediated hypertrophic growth of NRVMs as siRNA-mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). Additionally, HuR overexpression is sufficient to induce hypertrophic cell growth. To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT-luciferase reporter assay, we show an acute inhibition of NFAT transcriptional activity following pharmacological inhibition of HuR. In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq-p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.

  4. The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD through the MAPK pathway

    SciTech Connect

    Liu, Yanling; Xu, Sanpeng; Xiao, Fei; Xiong, Yan; Wang, Xiaojin; Gao, Sui; Yan, Weiming; Ning, Qin

    2010-05-28

    Fibrinogen-like protein 2 (FGL2)/fibroleukin has been reported to play a vital role in the pathogenesis of some critical inflammatory diseases by possessing immunomodulatory activity through the mediation of 'immune coagulation' and the regulation of maturation and proliferation of immune cells. We observed upregulated FGL2 expression in alveolar macrophages from peripheral lungs of chronic obstructive pulmonary disease (COPD) patients and found a correlation between FGL2 expression and increased macrophage activation markers (CD11b and CD14). The role of FGL2 in the activation of macrophages was confirmed by the detection of significantly decreased macrophage activation marker (CD11b, CD11c, and CD71) expression as well as the inhibition of cell migration and inflammatory cytokine (IL-8 and MMP-9) production in an LPS-induced FGL2 knockdown human monocytic leukemia cell line (THP-1). Increased FGL2 expression co-localized with upregulated phosphorylated p38 mitogen-activated protein kinase (p38-MAPK) in the lung tissues from COPD patients. Moreover, FGL2 knockdown in THP-1 cells significantly downregulated LPS-induced phosphorylation of p38-MAPK while upregulating phosphorylation of c-Jun N-terminal kinase (JNK). Thus, we demonstrate that FGL2 plays an important role in macrophage activation in the lungs of COPD patients through MAPK pathway modulation.

  5. Multiple MAPK cascades regulate the transcription of IME1, the master transcriptional activator of meiosis in Saccharomyces cerevisiae.

    PubMed

    Kahana-Edwin, Smadar; Stark, Michal; Kassir, Yona

    2013-01-01

    The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis. PMID:24236068

  6. Multiple MAPK Cascades Regulate the Transcription of IME1, the Master Transcriptional Activator of Meiosis in Saccharomyces cerevisiae

    PubMed Central

    Kahana-Edwin, Smadar; Stark, Michal; Kassir, Yona

    2013-01-01

    The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis. PMID:24236068

  7. Caffeine inhibits UV-mediated NF-kappaB activation in A2058 melanoma cells: an ATM-PKCdelta-p38 MAPK-dependent mechanism.

    PubMed

    Ravi, Dashnamoorthy; Muniyappa, Harish; Das, Kumuda C

    2008-01-01

    Mammalian ultraviolet (UV) radiation response is a gene induction cascade activated by several transcription factors, including NF-kappaB. Although NF-kappaB is induced by UV radiation, the signal transduction mechanism remains relatively unclear. In the present study, we show that UV-induced NF-kappaB activation is mediated by the activation of Ataxia telangiecia mutated (ATM) and protein kinase C (PKC). We also show that caffeine specifically inhibits UV-mediated NF-kappaB activation, but not TNFalpha-mediated NF-kappaB activation. In addition, our study shows that ATM, but not ATM-Rad3-related (ATR) or DNA-dependent protein kinase (DNA-PK) is involved in UV-induced NF-kappaB activation. Because SB203580 (a p38 MAPK inhibitor), or Calphostin C or rottlerin (PKC inhibitors) was able to inhibit UV-mediated NF-kappaB activation, we evaluated whether caffeine could inhibit p38 MAPK or PKC activity. Caffeine or rottlerin inhibited UV-induced phosphorylation of p38 MAPK, but not anisomycin-induced phosphorylation of p38 MAPK, suggesting that p38 MAPK is downstream of PKC. Additionally, caffeine could effectively inhibit UV-induced increases in PKC activity. Taken together, our study demonstrates that caffeine is a potent inhibitor of UV-induced NF-kappaB activation. Additionally, this inhibition occurs due to the inhibitory action of caffeine on ATM and PKC, resulting in the inhibition of p38 MAPK activation.

  8. Stress activated protein kinases, JNKs and p38 MAPK, are differentially activated in ganglia and heart of land snail Helix lucorum (L.) during seasonal hibernation and arousal.

    PubMed

    Michaelidis, Basile; Hatzikamari, Maria; Antoniou, Vassiliki; Anestis, Andreas; Lazou, Antigone

    2009-06-01

    The present work aimed to investigate the phosphorylation and hence activation of stress activated protein kinases, p38 MAPK and JNKs in the tissues of the snail Helix lucorum during seasonal hibernation. Snails were put in large glass boxes, which were placed outdoors so that they were exposed to natural conditions of light and temperature. Phosphorylation and hence activation of JNKs and p38 MAPK was determined in both heart and ganglia. Deep hibernation caused significant increases in the levels of the phosphorylated form of JNK and p38-MAPK in both heart and ganglia. Phosphorylation of JNK remained elevated in the ganglia or increased after a transient drop in the heart, when the snails were prepared for arousal. In addition, phosphorylation of p38-MAPK was further increased in the heart during this period. These data support the conclusion that MAPK signalling cascade might contribute in the physiological and biochemical remodelling in the tissues of land snails during hibernation and upon preparation for arousal.

  9. atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway

    SciTech Connect

    Yu Zengli . E-mail: yuzengli@263.net; Xing Ying . E-mail: xingy@zzu.edu.cn

    2006-08-15

    Our previous studies have shown that atRA treatment resulted in cell-cycle block and growth inhibition in mouse embryonic palatal mesenchymal (MEPM). In the current study, gestation day (GD) 13 MEPM cells were used to test the hypothesis that the growth inhibition by atRA is due to apoptosis. The effects of atRA on apoptosis were assessed by performing MTT assay, Cell Death Detection ELISA and flow cytometry, respectively. Data analysis confirmed that atRA treatment induced apoptosis-like cell death, as shown by decreased cell viability and increased fragmented DNA and sub-G1 fraction. atRA-induced apoptosis was associated with upregulation of bcl-2, translocation of bax protein to the mitochondria from the cytosol, activation of caspase-3 and cytochrome c release into cytosol. atRA-induced apoptosis was abrogated by z-DEVD-fmk, a caspase-3 specific inhibitor, and z-VAD-fmk, a general caspase inhibitor, suggesting that the atRA-induced cell death of MEPM cells occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, atRA treatment caused a strong and sustained activation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38), as well as an early but transient activation of extracellular signal-regulated kinase (ERK). Importantly, atRA-induced DNA fragmentation and capase-3 activation were prevented by pretreatment with the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB202190), but not by pretreatment with MEK inhibitor (U0126). From these results, we suggest that mitogen-activated protein kinase-dependent pathways is involved in the atRA-induced apoptosis of MEPM cells.

  10. Maternal Inheritance of Twist and Analysis of MAPK Activation in Embryos of the Polychaete Annelid Platynereis dumerilii

    PubMed Central

    Pfeifer, Kathrin; Schaub, Christoph; Domsch, Katrin; Dorresteijn, Adriaan; Wolfstetter, Georg

    2014-01-01

    In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a

  11. Apoptotic Effects of Cordycepin Through the Extrinsic Pathway and p38 MAPK Activation in Human Glioblastoma U87MG Cells.

    PubMed

    Baik, Ji-Sue; Mun, Seo-Won; Kim, Kyoung-Sook; Park, Shin-Ji; Yoon, Hyun-Kyoung; Kim, Dong-Hyun; Park, Min-Kyu; Kim, Cheorl-Ho; Lee, Young-Choon

    2016-02-01

    We first demonstrated that cordycepin inhibited cell growth and triggered apoptosis in U87MG cells with wild-type p53, but not in T98G cells with mutant-type p53. Western blot data revealed that the levels of procaspase-8, -3, and Bcl-2 were downregulated in cordycepintreated U87MG cells, whereas the levels of Fas, FasL, Bak, cleaved caspase-3, -8, and cleaved PARP were upregulated, indicating that cordycepin induces apoptosis by activating the death receptor-mediated pathway in U87MG cells. Cordycepin-induced apoptosis could be suppressed by only SB203580, a p38 MAPK-specific inhibitor. These results suggest that cordycepin triggered apoptosis in U87MG cells through p38 MAPK activation and inhibition of the Akt survival pathway. PMID:26597532

  12. Infectious bursal disease virus infection induces macrophage activation via p38 MAPK and NF-kappaB pathways.

    PubMed

    Khatri, Mahesh; Sharma, Jagdev M

    2006-06-01

    In the present study, we show that infection with infectious bursal disease virus (IBDV) causes activation of macrophages, the key cells involved in inflammatory and immune-regulatory functions. Exposure of cultured spleen macrophages (SM) from SPF chickens to IBDV resulted in the production of nitric oxide (NO). In addition, there was upregulation of mRNA expression of inducible nitric oxide synthase (iNOS), IL-8 and cyclooxygenase-2 (COX-2). The signal transduction pathways involved in macrophage activation were examined. The role of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) was tested by using specific pharmacological inhibitors. Addition of p38 MAPK inhibitor, SB-203580 and NF-kappaB inhibitor Bay 11-7082, suppressed IBDV-induced NO production and mRNA expression of iNOS, IL-8 and COX-2. The results suggest that IBDV uses cellular signal transduction machinery, in particular the p38 MAPK and NF-kappaB pathways, to elicit macrophage activation. The increased production of NO, IL-8 and COX-2 by macrophages may contribute to bursa inflammatory responses commonly seen during the acute IBDV infection.

  13. Induction of prostaglandin D2 through the p38 MAPK pathway is responsible for the antipruritic activity of sertaconazole nitrate.

    PubMed

    Kaur, Simarna; Sur, Runa; Liebel, Frank T; Southall, Michael D

    2010-10-01

    Prostaglandin D(2) (PGD(2)) is known to have antipruritic activity by suppressing histamine release. However, agents that can topically induce PGD(2) for itch relief are not well established. The antimycotic sertaconazole nitrate (STZ) has been shown to exhibit anti-itch properties; however, the mechanism for this activity has not been elucidated. STZ mitigated degranulation of RBL-2H3 (rat basophilic leukemia) mast cells induced by compound 48/80, a pruritogenic agent known to promote the release of histamine, and augmented PGD(2) production in mast cells and macrophages. Addition of exogenous PGD(2) abrogated compound 48/80-induced degranulation by acting through the prostanoid D receptor 1 (DP1). STZ induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in mast cells and a pharmacological inhibitor of p38 MAPK, SB203580, resulted in the attenuation of PGD(2) levels. Finally, in a murine model of pruritus, the scratching behavior induced by compound 48/80 was mitigated by topical application of STZ. This effect was reversed by the addition of the cyclooxygenase inhibitor, ibuprofen, or a DP1 receptor antagonist (MK0524). Collectively, these results suggest that STZ mediates its anti-itch effects by boosting the antipruritic agent, PGD(2), by the activation of the p38-MAPK pathway. This is the first report to demonstrate a promising approach to topically induce PGD(2) for improving pruritus. PMID:20505747

  14. Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma.

    PubMed

    Fowles, J S; Denton, C L; Gustafson, D L

    2015-09-01

    The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations. PMID:23745794

  15. YES oncogenic activity is specified by its SH4 domain and regulates RAS/MAPK signaling in colon carcinoma cells.

    PubMed

    Dubois, Fanny; Leroy, Cédric; Simon, Valérie; Benistant, Christine; Roche, Serge

    2015-01-01

    Members of the SRC family of tyrosine kinases (SFK) display important functions in human cancer, but their specific role in tumorigenesis remains unclear. We previously demonstrated that YES regulates a unique oncogenic signaling important for colorectal cancer (CRC) progression that is not shared with SRC. Here, we addressed the underlying mechanism involved in this process. We show that YES oncogenic signaling relies on palmitoylation of its SH4 domain that controls YES localization in cholesterol-enriched membrane micro-domains. Specifically, deletion of the palmitoylation site compromised YES transforming activity, while addition of a palmitoylation site in the SH4 domain of SRC was sufficient for SRC to restore the transforming properties of cells in which YES had been silenced. Subsequently, SILAC phosphoproteomic analysis revealed that micro-domain-associated cell adhesive components and receptor tyrosine kinases are major YES substrates. YES also phosphorylates upstream regulators of RAS/MAPK signaling, including EGFR, SHC and SHP2, which were not targeted by SRC due to the absence of palmitoylation. Accordingly, EGFR-induced MAPK activity was attenuated by YES down-regulation, while increased RAS activity significantly restored cell transformation that was lost upon YES silencing. Collectively, these results uncover a critical role for the SH4 domain in the specification of SFK oncogenic activity and a selective role for YES in the induction of RAS/MAPK signaling in CRC cells.

  16. 2-Methoxyestradiol induced Bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation.

    PubMed

    Min, Hongbo; Ghatnekar, Gautam S; Ghatnekar, Angela V; You, Xiaohong; Bu, Min; Guo, Xinyi; Bu, Shizhong; Shen, Bo; Huang, Qin

    2012-07-01

    Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2-Methoxyestradiol (2ME), an endogenous metabolite of 17-β-estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time- and dose-dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME-induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr(167) after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl-2-Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME-induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen-activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb. PMID:21769948

  17. Secretory pathway retention of mutant prion protein induces p38-MAPK activation and lethal disease in mice

    PubMed Central

    Puig, Berta; Altmeppen, Hermann C.; Ulbrich, Sarah; Linsenmeier, Luise; Krasemann, Susanne; Chakroun, Karima; Acevedo-Morantes, Claudia Y.; Wille, Holger; Tatzelt, Jörg; Glatzel, Markus

    2016-01-01

    Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrPC) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrPSc leading to prion diseases. We show that a deletion in the C-terminal domain of PrPC (PrPΔ214–229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrPC. Transgenic (Tg(PrPΔ214–229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214–229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice. PMID:27117504

  18. Maternal inflammation activated ROS-p38 MAPK predisposes offspring to heart damages caused by isoproterenol via augmenting ROS generation

    PubMed Central

    Zhang, Qi; Deng, Yafei; Lai, Wenjing; Guan, Xiao; Sun, Xiongshan; Han, Qi; Wang, Fangjie; Pan, Xiaodong; Ji, Yan; Luo, Hongqin; Huang, Pei; Tang, Yuan; Gu, Liangqi; Dan, Guorong; Yu, Jianhua; Namaka, Michael; Zhang, Jianxiang; Deng, Youcai; Li, Xiaohui

    2016-01-01

    Maternal inflammation contributes to the increased incidence of adult cardiovascular disease. The current study investigated the susceptibility of cardiac damage responding to isoproterenol (ISO) in adult offspring that underwent maternal inflammation (modeled by pregnant Sprague-Dawley rats with lipopolysaccharides (LPS) challenge). We found that 2 weeks of ISO treatment in adult offspring of LPS-treated mothers led to augmented heart damage, characterized by left-ventricular systolic dysfunction, cardiac hypertrophy and myocardial fibrosis. Mechanistically, prenatal exposure to LPS led to up-regulated expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, antioxidant enzymes, and p38 MAPK activity in left ventricular of adult offspring at resting state. ISO treatment exaggerated ROS generation, p38 MAPK activation but down-regulated reactive oxygen species (ROS) elimination capacity in the left ventricular of offspring from LPS-treated mothers, while antioxidant N-acetyl-L-cysteine (NAC) reversed these changes together with improved cardiac functions. The p38 inhibitor SB202190 alleviated the heart damage only via inhibiting the expression of NADPH oxidases. Collectively, our data demonstrated that prenatal inflammation programs pre-existed ROS activation in the heart tissue, which switches on the early process of oxidative damages on heart rapidly through a ROS-p38 MAPK-NADPH oxidase-ROS positive feedback loop in response to a myocardial hypertrophic challenge in adulthood. PMID:27443826

  19. OXIDATIVE STRESS PARTICIPATES IN ACUTE LUNG INJURY AND ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASES (MAPK) FOLLOWING AIR POLLUTION PARTICLE EXPOSURE (PM)

    EPA Science Inventory

    OXIDATIVE STRESS PARTICIPATES IN ACUTE LUNG INJURY AND ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASES (MAPK) FOLLOWING AIR POLLUTION PARTICLE EXPOSURE (PM). E S Roberts1, R Jaskot2, J Richards2, and K L Dreher2. 1College of Veterinary Medicine, NC State University, Raleigh, NC a...

  20. MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

    SciTech Connect

    Simões, Maylla Ronacher; Aguado, Andrea; Fiorim, Jonaína; Silveira, Edna Aparecida; Azevedo, Bruna Fernandes; Toscano, Cindy Medice; Zhenyukh, Olha; Briones, Ana María; Alonso, María Jesús; Vassallo, Dalton Valentim; Salaices, Mercedes

    2015-03-01

    Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100 g; subsequent doses: 0.125 μg/100 g, intramuscular, 30 days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20 μg/dL) were used. Lead blood levels of treated rats attained 21.7 ± 2.38 μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension. - Highlights: • Lead-exposure increases oxidative stress, COX-2 expression and vascular reactivity. • Lead exposure activates MAPK signaling pathway. • ROS and COX-2 activation by

  1. Thrombosis recanalization by paeoniflorin through the upregulation of urokinase-type plasminogen activator via the MAPK signaling pathway

    PubMed Central

    YE, SONGSHAN; MAO, BINGYU; YANG, LEI; FU, WEIYUN; HOU, JUNRAN

    2016-01-01

    Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen-activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme-linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6-keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D-dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti-thrombosis pathways. PMID:27082639

  2. Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

    PubMed Central

    Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

    2010-01-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

  3. p38 Mitogen-Activated Protein Kinase (MAPK) Increases Arginase Activity and Contributes to Endothelial Dysfunction in Corpora Cavernosa from Angiotensin-II Treated Mice

    PubMed Central

    Toque, Haroldo A.; Romero, Maritza J.; Tostes, Rita C.; Shatanawi, Alia; Chandra, Surabhi; Carneiro, Zidonia N.; Inscho, Edward W.; Webb, R. Clinton; Caldwell, Ruth B.; Caldwell, R. William

    2010-01-01

    Introduction Angiotensin II (AngII) activates p38 mitogen-activated protein kinase (MAPK) and elevates arginase activity in endothelial cells. Upregulation of arginase activity has been implicated in endothelial dysfunction by reducing NO bioavailability. However, signaling pathways activated by AngII in the penis are largely unknown. Aim We hypothesized that activation of p38 MAPK increases arginase activity and thus impairs penile vascular function in AngII-treated mice. Methods Male C57BL/6 mice were implanted with osmotic minipumps containing saline or AngII (42 μg/kg/h) for 14 days and co-treated with p38 MAPK inhibitor, SB 203580 (5 μg/kg/day), beginning 2 days before minipump implantation. Systolic blood pressure (SBP) was measured. Corpus cavernosum (CC) tissue was used for vascular functional studies and protein expression levels of p38 MAPK, arginase and constitutive NOS, and arginase activity. Main Outcome Measures Arginase expression and activity; expression of phospho-p38 MAPK, -eNOS and nNOS proteins; endothelium-dependent and nitrergic nerve-mediated relaxations were determined in CC from control and AngII-infused mice. Results AngII increased SBP (22%) and increased CC arginase activity and expression (~2-fold), and phosphorylated P38 MAPK levels (30%) over control. Treatment with SB 203580 prevented these effects. Endothelium-dependent NO-mediated relaxation to acetylcholine was significantly reduced by AngII and this effect was prevented by SB 203580 (P<0.01). AngII (2-week) did not alter nitrergic function. However, SB 203580 significantly increased nitrergic relaxation in both control and AngII tissue at lower frequencies. Maximum contractile responses for phenylephrine and electrical field stimulation were increased by AngII (56% and 171%, respectively), and attenuated by SB 203580 treated. AngII treatment also decreased eNOS phosphorylation at Ser-1177 compared to control. Treatment with SB 203580 prevented all these changes. Conclusion p38

  4. Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells.

    PubMed

    Zhong, Wu; Zhu, Haichuan; Sheng, Fugeng; Tian, Yonglu; Zhou, Jun; Chen, Yingyu; Li, Song; Lin, Jian

    2014-07-01

    Transition metal copper (Cu) can exist in oxidized or reduced states in cells, leading to cytotoxicity in cancer cells through oxidative stress. Recently, copper complexes are emerging as a new class of anticancer compounds. Here, we report that a novel anticancer copper complex (HYF127c/Cu) induces oxidative stress-dependent cell death in cancer cells. Further, transcriptional analysis revealed that oxidative stress elicits broad transcriptional changes of genes, in which autophagy-related genes are significantly changed in HYF127c/Cu-treated cells. Consistently, autophagy was induced in HYF127c/Cu-treated cells and inhibitors of autophagy promoted cell death induced by HYF127c/Cu. Further analysis identified that the MAPK11/12/13/14 (formerly known as p38 MAPK) pathway was also activated in HYF127c/Cu-treated cells. Meanwhile, the MAPK11/12/13/14 inhibitor SB203580 downregulated autophagy by inhibiting the transcription of the autophagy genes MAP1LC3B, BAG3, and HSPA1A, and promoted HYF127c/Cu-induced cell death. These data suggest that copper-induced oxidative stress will induce protective autophagy through transcriptional regulation of autophagy genes by activation of the MAPK11/12/13/14 pathway in HeLa cells.

  5. Dynamic ubiquitination of the mitogen-activated protein kinase kinase (MAPKK) Ste7 determines mitogen-activated protein kinase (MAPK) specificity.

    PubMed

    Hurst, Jillian H; Dohlman, Henrik G

    2013-06-28

    Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF(Cdc4) and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF(Cdc4) ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF(Cdc4) and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF(Cdc4) is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK. PMID:23645675

  6. Anti-Inflammatory Effects of α-Galactosylceramide Analogs in Activated Microglia: Involvement of the p38 MAPK Signaling Pathway

    PubMed Central

    Chung, Young Sun; Park, Seung Bum; Kim, Hee-Sun

    2014-01-01

    Microglial activation plays a pivotal role in the development and progression of neurodegenerative diseases. Thus, anti-inflammatory agents that control microglial activation can serve as potential therapeutic agents for neurodegenerative diseases. Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia. We performed biological evaluations of 25 α-GalCer analogs and observed an interesting preliminary structure-activity relationship in their inhibitory influence on NO release and TNF-α production in LPS-stimulated BV2 microglial cells. After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis. We confirmed that 4d and 4e regulate the expression of iNOS, COX-2, IL-1β, and IL-6 at the mRNA level and the expression of TNF-α at the post-transcriptional level. In addition, both 4d and 4e inhibited LPS-induced DNA binding activities of NF-κB and AP-1 and phosphorylation of p38 MAPK without affecting other MAP kinases. When we examined the anti-inflammatory effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation, we observed an identical inhibitory pattern as that of 4d and 4e, not only on NO and TNF-α production but also on the DNA binding activities of NF-κB and AP-1. Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities. PMID:24523867

  7. CyHV-2 ORF104 activates the p38 MAPK pathway.

    PubMed

    Du, Mi; Chen, Mingliang; Shen, Haifeng; Wang, Wei; Li, Zengpeng; Wang, Weiyi; Huang, Jianhui; Chen, Jianming

    2015-10-01

    Cyprinid herpesvirus 2 (CyHV-2) is the pathogen responsible for herpesviral hematopoietic necrosis disease, which causes huge losses on aquaculture. So far the studies of CyHV-2 mainly focus on the identification and detection of this virus, but little is known about the role of specific CyHV-2 genes in the infection process. Based on the genomic information, CyHV-2 ORF104 encodes a kinase-like protein, which is highly conserved among the three CyHVs. Our study was initiated to investigate the role of kinase-like protein ORF104 during virus infection. Subcellular localization study showed that ORF104 was mainly expressed in the nucleus in both human HEK293T and fish EPC cells. However, deletion of the putative nuclear localization signal of ORF104 (ORF104M) resulted in the cytoplasmic distribution in HEK293T. We then examined whether MAPKs were involved in the ORF104-mediated signaling pathway by overexpressing ORF104 and ORF104M in HEK293T. Overexpression of ORF104 and ORF104M resulted in the up-regulation of p38 phosphorylation, but not JNK or ERK, indicating that ORF104 specifically activates p38 signaling pathway. In vivo study showed that CyHV-2 infection enhanced p38 phosphorylation in gibel carp (Carassius auratus gibelio). Interestingly, p38 inhibitor SB203580 strongly reduced fish death caused by CyHV-2 infection. Therefore, our study for the first time reveals the function of ORF104 during CyHV-2 infection, indicating that ORF104 is a potential vaccine candidate for CyHV-2.

  8. Activation of MAPK Is Required for ROS Generation and Exocytosis in HMC-1 Cells Induced by Trichomonas vaginalis-Derived Secretory Products

    PubMed Central

    Narantsogt, Giimaa; Min, Arim; Nam, Young Hee; Lee, Young Ah; Kim, Kyeong Ah; Agvaandaram, Gurbadam; Dorjsuren, Temuulen; El-Benna, Jamel; Shin, Myeong Heon

    2015-01-01

    Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47phox in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling. PMID:26537039

  9. Activation of MAPK Is Required for ROS Generation and Exocytosis in HMC-1 Cells Induced by Trichomonas vaginalis-Derived Secretory Products.

    PubMed

    Narantsogt, Giimaa; Min, Arim; Nam, Young Hee; Lee, Young Ah; Kim, Kyeong Ah; Agvaandaram, Gurbadam; Dorjsuren, Temuulen; El-Benna, Jamel; Shin, Myeong Heon

    2015-10-01

    Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47(phox) in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling.

  10. Activation of p21ras/MAPK signal transduction molecules decreases with age in mitogen-stimulated T cells from rats.

    PubMed

    Pahlavani, M A; Harris, M D; Richardson, A

    1998-04-10

    Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4-6 months) and old (24-26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21ras activities. The time course of induction of MAPK/JNK and p21ras activities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that the in vitro kinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.

  11. P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

    PubMed

    Nerurkar, S S; Olzinski, A R; Frazier, K S; Mirabile, R C; O'Brien, S P; Jing, J; Rajagopalan, D; Yue, T L; Willette, R N

    2007-01-01

    The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression

  12. Oxidized High-Density Lipoprotein Impairs Endothelial Progenitor Cells' Function by Activation of CD36-MAPK-TSP-1 Pathways

    PubMed Central

    Wu, Jianxiang; He, Zhiqing; Gao, Xiang; Wu, Feng; Ding, Ru; Ren, Yusheng; Jiang, Qijun; Fan, Min

    2015-01-01

    Abstract Aims: High-density lipoprotein (HDL) levels inversely correlate with cardiovascular events due to the protective effects on vascular wall and stem cells, which are susceptible to oxidative modifications and then lead to potential pro-atherosclerotic effects. We proposed that oxidized HDL (ox-HDL) might lead to endothelial progenitor cells (EPCs) dysfunction and investigated underlying mechanisms. Results: ox-HDL was shown to increase apoptosis and intracellular reactive oxygen species levels, but to reduce migration, angiogenesis, and cholesterol efflux of EPCs in a dose-dependent manner. p38 mitogen-activated protein kinase (MAPK) and NF-κB were activated after ox-HDL stimulation, which also upregulated thrombospondin-1 (TSP-1) expression without affecting vascular endothelial growth factor. Effects caused by ox-HDL could be significantly attenuated by pretreatment with short hairpin RNA-mediated CD36 knockdown or probucol. Data of in vivo experiments and the inverse correlation of ox-HDL and circulating EPC numbers among patients with coronary artery diseases (CAD) or CAD and type 2 diabetes also supported it. Meanwhile, HDL separated from such patients could significantly increase cultured EPC's caspase 3 activity, further supporting our proposal. Innovation: This is the most complete study to date of how ox-HDL would impair EPCs function, which was involved with activation of CD36-p38 MAPK-TSP-1 pathways and proved by not only the inverse relationship between ox-HDL and circulating EPCs in clinic but also pro-apoptotic effects of HDL separated from patients' serum. Conclusion: Activation of CD36-p38 MAPK-TSP-1 pathways contributes to the pathological effects of ox-HDL on EPCs' dysfunction, which might be one of the potential etiological factors responsible for the disturbed neovascularization in chronic ischemic disease. Antioxid. Redox Signal. 22, 308–324. PMID:25313537

  13. Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- and Arrestin-dependent in Neurons and Astrocytes*

    PubMed Central

    Bruchas, Michael R.; Macey, Tara A.; Lowe, Janet D.; Chavkin, Charles

    2007-01-01

    AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (γ-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism. PMID:16648139

  14. Activation of neutral-sphingomyelinase, MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

    PubMed Central

    2014-01-01

    Background Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells. Results C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869. Conclusions Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma. PMID:24997497

  15. Distinct docking mechanisms mediate interactions between the Msg5 phosphatase and mating or cell integrity mitogen-activated protein kinases (MAPKs) in Saccharomyces cerevisiae.

    PubMed

    Palacios, Lorena; Dickinson, Robin J; Sacristán-Reviriego, Almudena; Didmon, Mark P; Marín, María José; Martín, Humberto; Keyse, Stephen M; Molina, María

    2011-12-01

    MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif ((102)IYT(104)) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274-373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity. PMID:22006927

  16. Polycyclic aromatic hydrocarbon (PAH)-mediated upregulation of hepatic microRNA-181 family promotes cancer cell migration by targeting MAPK phosphatase-5, regulating the activation of p38 MAPK

    SciTech Connect

    Song, Mi-Kyung; Park, Yong-Keun; Ryu, Jae-Chun

    2013-11-15

    Growing evidence indicates that changes in microRNA (miRNA) expression in cancer induced by chemical carcinogens play an important role in cancer development and progression by regulating related genes. However, the mechanisms underlying miRNA involvement in hepatocarcinogenesis induced by polycyclic aromatic hydrocarbons (PAHs) remain unclear. Thus, the identification of aberrant miRNA expression during PAH-induced cancer cell migration will lead to a better understanding of the substantial role of miRNAs in cancer progression. In the present study, miRNA expression profiling showed significant upregulation of miR-181a, -181b, and -181d in human hepatocellular carcinoma cells (HepG2 line) exposed to benzo[a]anthracene (BA) and benzo[k]fluoranthene (BF). MAPK phosphatase-5 (MKP-5), a validated miR-181 target that deactivates MAPKs, was markedly suppressed while phosphorylation of p38 MAPK was increased after BA and BF exposure. The migration of HepG2 cells, observed using the scratch wound-healing assay, also increased in a dose-dependent manner. Depletion of miR-181 family members by miRNA inhibitors enhanced the expression of MKP-5 and suppressed the phosphorylation of p38 MAPK. Furthermore, the depletion of the miR-181 family inhibited cancer cell migration. Based on these results, we conclude that the miR-181 family plays a critical role in PAH-induced hepatocarcinogenesis by targeting MKP-5, resulting in the regulation of p38 MAPK activation. - Highlights: • We found significant upregulation of miR-181 family in HCC exposed to BA and BF. • We identified the MKP-5 as a putative target of miR-181 family. • MKP-5 was suppressed while p-P38 was increased after BA and BF exposure. • The migration of HepG2 cells increased in a dose-dependent manner.

  17. Inhibitory effect of panduratin A on UV-induced activation of mitogen-activated protein kinases (MAPKs) in dermal fibroblast cells.

    PubMed

    Shim, Jae-Seok; Kwon, Yi-Young; Han, Young-Sun; Hwang, Jae-Kwan

    2008-10-01

    Exposure of the skin to ultraviolet (UV) induces photoaging associated with up-regulated matrix metalloproteinases (MMPs) activities and decreased collagen synthesis. We previously reported that panduratin A, a chalcone compound isolated from KAEMPFERIA PANDURATA Roxb ., decreased MMP-1 expression in UV-irradiated human skin fibroblasts. Here, we have investigated the effect of panduratin A on UV-induced activation of mitogen-activated protein kinases (MAPKs) signaling modules such as extracellular-regulated protein kinase (ERK), Jun-N-terminal kinase (JNK) and p38 kinase. Treatment with panduratin A in the range of 0.001 - 0.1 microM significantly inhibited UV-induced ERK, JNK and p38 activation. Moreover, inhibition of ERK, JNK and p38 by panduratin A resulted in decreased c-Fos expression and c-Jun phosphorylation induced by UV, which led to inhibition of activator protein-1 (AP-1) DNA binding activity. Panduratin A showed stronger activity than epigallocatechin 3- O-gallate (EGCG) known as a natural anti-aging agent. The results suggest that panduratin A can down-regulate UV-induced MMP-1 expression by inhibiting the MAPKs pathways and AP-1 activation. AP-1:activator protein-1 EGCG:epigallocatechin 3- O-gallate ERK:extracellular-regulated protein kinase JNK:c-Jun N-terminal kinase MAPK:mitogen-activated protein kinase MMP:matrix metalloproteinase UV:ultraviolet.

  18. MAPK Cascades in Guard Cell Signal Transduction

    PubMed Central

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M.

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  19. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions.

  20. MAPK Cascades in Guard Cell Signal Transduction.

    PubMed

    Lee, Yuree; Kim, Yun Ju; Kim, Myung-Hee; Kwak, June M

    2016-01-01

    Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions. PMID:26904052

  1. Canonical and alternative MAPK signaling.

    PubMed

    Pimienta, Genaro; Pascual, Jaime

    2007-11-01

    The archetype of MAPK cascade activation is somewhat challenged by the most recent discovery of unexpected phosphorylation patterns, alternative activation mechanisms and sub-cellular localization, in various members of this protein kinase family. In particular, activation by autophosphorylation pathways has now been described for the three best understood MAPK subgroups: ERK1/2; JNK1/2 and p38 alpha/beta. Also, a form of dosage compensation between homologs has been shown to occur in the case of ERK1/2 and JNK1/2. In this paper we summarize the MAPK activation pathway, with an emphasis on non-canonical examples. We use this information to propose a model for MAPK signal transduction that considers a cross-talk between MAPKs with different activation loop sequence motifs and unique C-terminal extensions. We highlight the occurrence of non-canonical substrate specificity during MAPK auto-activation, in strong connection with MAPK homo- and hetero-dimerization events.

  2. Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

    SciTech Connect

    Song, Xiulong Wei, Zhengxi; Shaikh, Zahir A.

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  3. Formin-induced actin cables are required for polarized recruitment of the Ste5 scaffold and high level activation of MAPK Fus3.

    PubMed

    Qi, Maosong; Elion, Elaine A

    2005-07-01

    Little is known about how a mitogen-activated protein kinase (MAPK) cascade is targeted to specific sites at the plasma membrane during receptor stimulation. In budding yeast, the Ste5 scaffold is recruited to a receptor-coupled G protein during mating pheromone stimulation, allowing the tethered MAPK cascade to be activated by Ste20, a Cdc42-anchored kinase. Here we show that stable recruitment of Ste5 at cortical sites requires the formin Bni1, Bni1-induced actin cables, Rho1 and Myo2. Rho1 directs recruitment of Bni1 via the Rho-binding domain, and Bni1 mediates localization of Ste5 through actin cables and Myo2, which co-immunoprecipitates with Ste5 during receptor stimulation. Bni1 is also required for polarized recruitment and full activation of MAPK Fus3, which must bind Ste5 to be activated, and polarized recruitment of Cdc24, the guanine exchange factor that binds Ste5 and promotes its recruitment to the G protein. In contrast, Bni1 is not important for activation of MAPK Kss1, which can be activated while not bound to Ste5 and does not accumulate at cortical sites. These findings reveal that Bni1 mediates the formation of a Ste5 scaffold/Fus3 MAPK signaling complex at polarized sites, and suggests that a pool of Ste5 may translocate along formin-induced actin cables to the cell cortex. PMID:15961405

  4. An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes

    PubMed Central

    Sun, Ming-Ju; Zhu, Shuai; Li, You-Wei; Lin, Juan; Gong, Shuai; Jiao, Guang-Zhong; Chen, Fei; Tan, Jing-He

    2016-01-01

    The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1–2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1–2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3–6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes. PMID:27009903

  5. Advanced Glycation End Products Impair Glucose-Stimulated Insulin Secretion of a Pancreatic β-Cell Line INS-1-3 by Disturbance of Microtubule Cytoskeleton via p38/MAPK Activation.

    PubMed

    You, Jia; Wang, Zai; Xu, Shiqing; Zhang, Wenjian; Fang, Qing; Liu, Honglin; Peng, Liang; Deng, Tingting; Lou, Jinning

    2016-01-01

    Advanced glycation end products (AGEs) are believed to be involved in diverse complications of diabetes mellitus. Overexposure to AGEs of pancreatic β-cells leads to decreased insulin secretion and cell apoptosis. Here, to understand the cytotoxicity of AGEs to pancreatic β-cells, we used INS-1-3 cells as a β-cell model to address this question, which was a subclone of INS-1 cells and exhibited high level of insulin expression and high sensitivity to glucose stimulation. Exposed to large dose of AGEs, even though more insulin was synthesized, its secretion was significantly reduced from INS-1-3 cells. Further, AGEs treatment led to a time-dependent increase of depolymerized microtubules, which was accompanied by an increase of activated p38/MAPK in INS-1-3 cells. Pharmacological inhibition of p38/MAPK by SB202190 reversed microtubule depolymerization to a stabilized polymerization status but could not rescue the reduction of insulin release caused by AGEs. Taken together, these results suggest a novel role of AGEs-induced impairment of insulin secretion, which is partially due to a disturbance of microtubule dynamics that resulted from an activation of the p38/MAPK pathway. PMID:27635403

  6. Advanced Glycation End Products Impair Glucose-Stimulated Insulin Secretion of a Pancreatic β-Cell Line INS-1-3 by Disturbance of Microtubule Cytoskeleton via p38/MAPK Activation

    PubMed Central

    You, Jia; Xu, Shiqing; Zhang, Wenjian; Fang, Qing; Liu, Honglin; Peng, Liang; Deng, Tingting

    2016-01-01

    Advanced glycation end products (AGEs) are believed to be involved in diverse complications of diabetes mellitus. Overexposure to AGEs of pancreatic β-cells leads to decreased insulin secretion and cell apoptosis. Here, to understand the cytotoxicity of AGEs to pancreatic β-cells, we used INS-1-3 cells as a β-cell model to address this question, which was a subclone of INS-1 cells and exhibited high level of insulin expression and high sensitivity to glucose stimulation. Exposed to large dose of AGEs, even though more insulin was synthesized, its secretion was significantly reduced from INS-1-3 cells. Further, AGEs treatment led to a time-dependent increase of depolymerized microtubules, which was accompanied by an increase of activated p38/MAPK in INS-1-3 cells. Pharmacological inhibition of p38/MAPK by SB202190 reversed microtubule depolymerization to a stabilized polymerization status but could not rescue the reduction of insulin release caused by AGEs. Taken together, these results suggest a novel role of AGEs-induced impairment of insulin secretion, which is partially due to a disturbance of microtubule dynamics that resulted from an activation of the p38/MAPK pathway.

  7. Advanced Glycation End Products Impair Glucose-Stimulated Insulin Secretion of a Pancreatic β-Cell Line INS-1-3 by Disturbance of Microtubule Cytoskeleton via p38/MAPK Activation

    PubMed Central

    You, Jia; Xu, Shiqing; Zhang, Wenjian; Fang, Qing; Liu, Honglin; Peng, Liang; Deng, Tingting

    2016-01-01

    Advanced glycation end products (AGEs) are believed to be involved in diverse complications of diabetes mellitus. Overexposure to AGEs of pancreatic β-cells leads to decreased insulin secretion and cell apoptosis. Here, to understand the cytotoxicity of AGEs to pancreatic β-cells, we used INS-1-3 cells as a β-cell model to address this question, which was a subclone of INS-1 cells and exhibited high level of insulin expression and high sensitivity to glucose stimulation. Exposed to large dose of AGEs, even though more insulin was synthesized, its secretion was significantly reduced from INS-1-3 cells. Further, AGEs treatment led to a time-dependent increase of depolymerized microtubules, which was accompanied by an increase of activated p38/MAPK in INS-1-3 cells. Pharmacological inhibition of p38/MAPK by SB202190 reversed microtubule depolymerization to a stabilized polymerization status but could not rescue the reduction of insulin release caused by AGEs. Taken together, these results suggest a novel role of AGEs-induced impairment of insulin secretion, which is partially due to a disturbance of microtubule dynamics that resulted from an activation of the p38/MAPK pathway. PMID:27635403

  8. Glycosphingolipid synthesis inhibitor represses cytokine-induced activation of the Ras-MAPK pathway in embryonic neural precursor cells.

    PubMed

    Yanagisawa, Makoto; Nakamura, Kazuo; Taga, Tetsuya

    2005-09-01

    Neuronal and glial cells in the central nervous system are generated from common neural precursor cells during development. To evaluate the functions of glycosphingolipids (GSLs) in neural precursor cells, neuroepithelial cells (NECs) were prepared from mouse embryos (E14.5), and the effects of an inhibitor of glucosylceramide synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), on NECs was investigated. In PDMP-treated NECs, the expression of GD3, a major ganglioside of NECs, disappeared. We found that basic fibroblast growth factor (bFGF)-induced proliferation and extracellular signal-regulated kinase (ERK) activation were repressed in PDMP-treated NECs. Leukemia inhibitory factor (LIF)-induced ERK activation was also abolished in PDMP-treated NECs, suggesting that PDMP specifically represses the Ras-MAPK pathway. bFGF-induced activation of the Ras-MAPK pathway in NECs is dependent on GSL-enriched microdomains, lipid rafts. The organization of lipid rafts and the distribution of Ras and Grb2-SOS in the microdomains were not affected. However, Ras activation was repressed in PDMP-treated NECs. In PDMP-treated NECs, some neuronal genes were up-regulated and glial genes were down-regulated. These results suggest that GSLs might be involved in the proliferation, survival, signal transduction and differentiation of NECs.

  9. TNF-α-induced depressive-like phenotype and p38(MAPK) activation are abolished by ascorbic acid treatment.

    PubMed

    Moretti, Morgana; Budni, Josiane; Freitas, Andiara Espíndola; Neis, Vivian Binder; Ribeiro, Camille Mertins; de Oliveira Balen, Grasiela; Rieger, Débora Kurrle; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

    2015-06-01

    We investigated the effects of ascorbic acid on depressive-like behavior induced by tumor necrosis factor (TNF-α) in mice. Additionally, we examined the effects of combined administration of ascorbic acid and antidepressants, MK-801 and 7-nitroindazole in mice exposed or not to TNF-α and the capacity of TNF-α and ascorbic acid to modulate hippocampal and cerebrocortical phosphorylation of extracellular signal-regulated kinase (ERK), p38(MAPK) and c-Jun N-terminal kinase (JNK). In control animals, ascorbic acid reduced the immobility time in the tail suspension test (TST). Unilateral intracerebroventricular administration of TNF-α produced a depressive-like behavior in the TST, and the treatment with ascorbic acid prevented this effect. Sub-effective dose of ascorbic acid combined with sub-effective doses of fluoxetine, imipramine, bupropion, MK-801 or 7-nitroindazole produced a synergistic antidepressant-like effect in mice exposed or not to TNF-α. No treatment caused significant alterations in the locomotor activity of mice. Administration of TNF-α increased the phosphorylation of p38(MAPK) in hippocampus and cerebral cortex, and the treatment with ascorbic acid prevented this effect. Ascorbic acid increased phosphorylation of ERK1 in the hippocampus of saline- and TNF-α-treated animals, however it did not produce alterations in the cerebral cortex. No effects on phosphorylation of ERK2 or JNK were found. The observed effect of ascorbic acid seems to be associated, at least partially, with a reduced p38(MAPK) phosphorylation, activation of the monoaminergic systems as well as inhibition of N-methyl-D-aspartate (NMDA) receptors and nitric oxide (NO) synthesis.

  10. The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

    PubMed

    Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

    2012-06-01

    The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. PMID:22215152

  11. A novel cysteine-rich neurotrophic factor in Aplysia facilitates growth, MAPK activation, and long-term synaptic facilitation.

    PubMed

    Pu, Lu; Kopec, Ashley M; Boyle, Heather D; Carew, Thomas J

    2014-04-01

    Neurotrophins are critically involved in developmental processes such as neuronal cell survival, growth, and differentiation, as well as in adult synaptic plasticity contributing to learning and memory. Our previous studies examining neurotrophins and memory formation in Aplysia showed that a TrkB ligand is required for MAPK activation, long-term synaptic facilitation (LTF), and long-term memory (LTM) for sensitization. These studies indicate that neurotrophin-like molecules in Aplysia can act as key elements in a functionally conserved TrkB signaling pathway. Here we report that we have cloned and characterized a novel neurotrophic factor, Aplysia cysteine-rich neurotrophic factor (apCRNF), which shares classical structural and functional characteristics with mammalian neurotrophins. We show that apCRNF (1) is highly enriched in the CNS, (2) enhances neurite elongation and branching, (3) interacts with mammalian TrkB and p75(NTR), (4) is released from Aplysia CNS in an activity-dependent fashion, (5) facilitates MAPK activation in a tyrosine kinase dependent manner in response to sensitizing stimuli, and (6) facilitates the induction of LTF. These results show that apCRNF is a native neurotrophic factor in Aplysia that can engage the molecular and synaptic mechanisms underlying memory formation.

  12. Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin, and CaMK-IV activities in rat T cells.

    PubMed

    Pahlavani, M A; Vargas, D M

    2000-02-01

    The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.

  13. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.

    PubMed

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    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 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF(WT) tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

  14. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

    PubMed Central

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    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 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAFWT tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.09976.001 PMID:26882073

  15. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway.

    PubMed

    Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

    2015-09-28

    Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue.

  16. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway

    PubMed Central

    Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

    2015-01-01

    Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue. PMID:26412401

  17. MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor.

    PubMed

    Chakraborty, Arup R; Robey, Robert W; Luchenko, Victoria L; Zhan, Zhirong; Piekarz, Richard L; Gillet, Jean-Pierre; Kossenkov, Andrew V; Wilkerson, Julia; Showe, Louise C; Gottesman, Michael M; Collie, Nathan L; Bates, Susan E

    2013-05-16

    To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

  18. Antioxidant effect of imperatorin from Angelica dahurica in hypertension via inhibiting NADPH oxidase activation and MAPK pathway.

    PubMed

    Cao, Yanjun; Zhang, Yanmin; Wang, Nan; He, Langchong

    2014-08-01

    Imperatorin (IMP) is an active furocoumarin in the traditional Chinese medicine Angelica dahurica and has been demonstrated to have vasodilatory activity. In the present study, we investigated the effect of IMP on blood pressure (BP) and antioxidant effects in spontaneously hypertensive rats (SHR) and human embryonic kidney 293 cells. SHR were administered IMP (6.25, 12.5, and 25 mg/kg/d) or tempol (18 mg/kg/d) daily by gavage for 12 weeks. Thiobarbituric acid-reactive substances, proteinuria levels, and superoxide dismutase activity were evaluated with commercial kits. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits of the renal cortical tissues were determined by reverse transcriptase polymerase chain reaction and Western blot. Twenty-four hour urinary 8-Iso-prostaglandin F2α was measured by enzyme linked immunosorbent assay. Systolic BP and diastolic BP were significantly reduced by treatment with IMP (6.25, 12.5, and 25 mg/kg/d) in SHR. Meanwhile, we found that renal cortical superoxide dismutase activities were significantly increased in IMP-treated groups. Renal cortical and urinary thiobarbituric acid-reactive substances' levels, the 24-hour urinary excretion of 8-Iso-prostaglandin F2α, and proteinuria in the IMP-treated group, were lower than SHR group. After that, we found the messenger RNA expressions and protein levels of NADPH oxidase subunits were markedly reduced after IMP treated in SHR. IMP also reduced the phosphorylation of protein kinase B, extracellular signal-regulated kinase1/2, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase in renal cortical in SHR. In addition, H2O2-induced ROS production in human embryonic kidney 293 cells was markedly attenuated by IMP. H2O2-induced activation of MAPK, protein kinase B, and expression of NADPH oxidase were also attenuated by pretreatment of IMP. In summary, IMP showed antihypertensive effect via prevention of renal injury not only by reducing NADPH

  19. p38 mitogen-activated protein kinase (p38 MAPK)-mediated autoimmunity: lessons to learn from ANCA vasculitis and pemphigus vulgaris.

    PubMed

    Mavropoulos, Athanasios; Orfanidou, Timoklia; Liaskos, Christos; Smyk, Daniel S; Billinis, Charalambos; Blank, Miri; Rigopoulou, Eirini I; Bogdanos, Dimitrios P

    2013-03-01

    Evidence is beginning to accumulate that p38 mitogen activated protein kinase (p38 MAPK) signaling pathway plays an important role in the regulation of cellular and humoral autoimmune responses. The exact mechanisms and the degree by which the p38 MAPK pathway participates in the immune-mediated induction of diseases have started to emerge. This review discusses the recent advances in the molecular dissection of the p38 MAPK pathway and the findings generated by reports investigating its role in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and autoimmune hepatitis. Application of newly-developed protocols based on sensitive flow cytometric detection has proven to be a useful tool in the investigation of the phosphorylation of p38 MAPK within different peripheral blood mononuclear cell populations and may help us to better understand the enigmatic role of this signaling cascade in the induction of autoimmunity as well as its role in immunosuppressive-induced remission. Special attention is paid to reported data proposing a specific role for autoantibody-induced activation of p38 MAPK-mediated immunopathology in the pathogenesis of autoimmune blistering diseases and anti-neutrophilic antibody-mediated vasculitides.

  20. MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway.

    PubMed

    Wu, Xiang-Song; Wang, Xu-An; Wu, Wen-Guang; Hu, Yun-Ping; Li, Mao-Lan; Ding, Qian; Weng, Hao; Shu, Yi-Jun; Liu, Tian-Yu; Jiang, Lin; Cao, Yang; Bao, Run-Fa; Mu, Jia-Sheng; Tan, Zhu-Jun; Tao, Feng; Liu, Ying-Bin

    2014-06-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is associated with metastasis and is an independent prognostic factor for lung cancer. Recent studies have demonstrated that MALAT1 plays an important role in other malignancies. However, little is known about the role of MALAT1 in gallbladder carcinoma (GBC), which is the most common cancer of the biliary tract and has an extremely poor prognosis. In this study, we focused on the expression, biological functions and mechanism of MALAT1 in GBC and found that MALAT1 was significantly upregulated in GBC tissues compared with corresponding non-cancerous tissues. Knockdown of MALAT1 in GBC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the GBC cells both in vitro and in vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC cell lines after MALAT1 knockdown. These results indicated that MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway.

  1. Tumor suppressor gene ING3 induces cardiomyocyte hypertrophy via inhibition of AMPK and activation of p38 MAPK signaling.

    PubMed

    Wang, Jiaojiao; Liu, Zhiping; Feng, Xiaojun; Gao, Si; Xu, Suowen; Liu, Peiqing

    2014-11-15

    Cardiac hypertrophy, an adaptive growth process that occurs in response to various pathophysiological stimuli, constitutes an important risk factor for the development of heart failure. However, the molecular mechanisms that regulate this cardiac growth response are not completely understood. Here we revealed that ING3 (inhibitor of growth family, member 3), a type II tumor suppressor, plays a critical role in the regulation of cardiac hypertrophy. ING3 expression was present in relatively high abundance in the heart, and was prominently upregulated in hypertrophic agonists angiotensin II (Ang II), phenylephrine (PE), or isoproterenol (ISO)-stimulated cardiomyocytes and in hearts of rat undergoing abdominal aortic constriction (AAC) surgery. In cardiomyocytes, overexpression of ING3 caused an increase in ANP, BNP and β-MHC mRNA levels and cell surface area, while depletion of ING3 attenuated PE-induced cardiomyocyte hypertrophy. Mechanistically, we have demonstrated that overexpression of ING3 could inactivate the AMPK and activate the canonical p38 MAPK signaling. Remarkably, AMPK agonist AICAR or p38 MAPK inhibitor SB203580 abrogated ING3-induced hypertrophic response in cardiomyocytes. In summary, our data disclose a novel role of ING3 as an inducer of pathological cardiac hypertrophy, suggesting that silencing of ING3 may be explored as a potential therapeutic target in preventing cardiac hypertrophy.

  2. The Chromone Alkaloid, Rohitukine, Affords Anti-Cancer Activity via Modulating Apoptosis Pathways in A549 Cell Line and Yeast Mitogen Activated Protein Kinase (MAPK) Pathway

    PubMed Central

    Safia; Kamil, Mohd; Jadiya, Pooja; Sheikh, Saba; Haque, Ejazul; Nazir, Aamir; Lakshmi, Vijai; Mir, Snober S.

    2015-01-01

    The field of cancer research and treatment has made significant progress, yet we are far from having completely safe, efficient and specific therapies that target cancer cells and spare the healthy tissues. Natural compounds may reduce the problems related to cancer treatment. Currently, many plant products are being used to treat cancer. In this study, Rohitukine, a natural occurring chromone alkaloid extracted from Dysoxylum binectariferum, was investigated for cytotoxic properties against budding yeast as well as against lung cancer (A549) cells. We endeavored to specifically study Rohitukine in S. cerevisiae in the context of MAPK pathways as yeast probably represents the experimental model where the organization and regulation of MAPK pathways are best understood. MAPK are evolutionarily conserved protein kinases that transfer extracellular signals to the machinery controlling essential cellular processes like growth, migration, differentiation, cell division and apoptosis. We aimed at carrying out hypothesis driven studies towards targeting the important network of cellular communication, a critical process that gets awry in cancer. Employing mutant strains of genetic model system Saccharomyces cerevisiae. S. cerevisiae encodes five MAPKs involved in control of distinct cellular responses such as growth, differentiation, migration and apoptosis. Our study involves gene knockouts of Slt2 and Hog1 which are functional homologs of human ERK5 and mammalian p38 MAPK, respectively. We performed cytotoxicity assay to evaluate the effect of Rohitukine on cell viability and also determined the effects of drug on generation of reactive oxygen species, induction of apoptosis and expression of Slt2 and Hog1 gene at mRNA level in the presence of drug. The results of this study show a differential effect in the activity of drug between the WT, Slt2 and Hog1 gene deletion strain indicating involvement of MAPK pathway. Further, we investigated Rohitukine induced cytotoxic

  3. Abrogation of NF-κB signaling in human neutrophils induces neutrophil survival through sustained p38-MAPK activation.

    PubMed

    Langereis, Jeroen D; Raaijmakers, Hanneke A J A; Ulfman, Laurien H; Koenderman, Leo

    2010-10-01

    NF-κB, an important transcription factor in the regulation of cellular inflammation, is one of the prime targets for novel anti-inflammatory therapeutics. Nowadays, anti-inflammatory therapies rely mostly on steroids, which among other effects, inhibit NF-κB activity. However, steroids have only limited efficacy in the treatment on neutrophil-driven diseases, such as COPD. Human neutrophils play an important role in the pathogenesis of COPD, and clearance of these cells by apoptosis is an effective pathway for resolution of inflammation. In this study, we tested the hypothesis that modulation of the NF-κB pathway in human neutrophils affects survival. Importantly, the pharmacological NF-κB inhibitor Bay 11-7082 inhibited NF-κB signaling in human neutrophils as expected. However, we found that complete inhibition of NF-κB activity with 10 μM Bay 11-7082 prolonged neutrophil survival significantly, which was not observed with inhibitors for other signaling pathways. Bay 11-7082-induced neutrophil survival was dependent on p38-MAPK kinase activity, as the p38 kinase activity inhibitor SB203580 abrogated this response completely. Bay 11-7082 induced rapid and sustained p38 activation that correlated with inhibited NF-κB signaling and prolonged neutrophil survival. The precise role of NF-κB in regulation of p38-MAPK activation remains to be established. Under these conditions of survival, the stability of Bcl-xL but not Mcl-1 was enhanced. Although inhibition of NF-κB leads to down-regulation of inflammatory genes in many cell types, our results illustrate that interference with basal NF-κB signaling in neutrophils as a drug target should be used with caution.

  4. 17β-Estradiol induces odontoblastic differentiation via activation of the c-Src/MAPK pathway in human dental pulp cells.

    PubMed

    Woo, Su Mi; Seong, Kyung Joo; Oh, Sang Jin; Park, Hong Ju; Kim, Sun Hun; Kim, Won Jae; Jung, Ji Yeon

    2015-12-01

    The present study is aimed at investigating the effects of the exogenous estrogen 17β-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.

  5. The Conserved MAPK Site in E(spl)-M8, an Effector of Drosophila Notch Signaling, Controls Repressor Activity during Eye Development

    PubMed Central

    Bandyopadhyay, Mohna; Bishop, Clifton P.

    2016-01-01

    The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to ‘cis’-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader

  6. TACE release of TNF-α mediates mechanotransduction-induced activation of p38 MAPK and myogenesis

    PubMed Central

    Zhan, Mei; Jin, Bingwen; Chen, Shuen-Ei; Reecy, James M.; Li, Yi-Ping

    2011-01-01

    Summary Skeletal muscle responds to mechanical stimulation by activating p38 MAPK, a key signal for myogenesis. However, the mechanotransduction mechanism that activates p38 is unknown. Here we show that mechanical stimulation of myoblasts activates p38 and myogenesis through stimulating TNF-α release by TNF-α converting enzyme (TACE). In C2C12 or mouse primary myoblasts cultured in growth medium, static stretch activated p38 along with ERK1/2, JNK and AKT. Disrupting TNF-α signaling by TNF-α-neutralizing antibody or knocking out TNF-α receptors blocked stretch activation of p38, but not ERK1/2, JNK or AKT. Stretch also activated differentiation markers MEF2C, myogenin, p21 and myosin heavy chain in a TNF-α- and p38-dependent manner. Stretch stimulated the cleavage activity of TACE. Conversely, TACE inhibitor TAPI or TACE siRNA abolished stretch activation of p38. In addition, conditioned medium from stretched myoblast cultures activated p38 in unstretched myoblasts, which required TACE activity in the donor myoblasts, and TNF-α receptors in the recipient myoblasts. These results indicate that posttranscriptional activation of TACE mediates the mechanotransduction that activates p38-dependent myogenesis via the release of TNF-α. PMID:17264149

  7. Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells

    SciTech Connect

    Cheon, Soyoung; Song, Seok Bean; Jung, Minkyung; Park, Yoorim; Bang, Jung-Wook; Kim, Tae Sung; Park, Hyunjeong; Kim, Cherl-hyun; Yang, Yool-hee; Bang, Sa Ik; Cho, Daeho

    2008-09-12

    Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-{gamma} inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-{gamma} production, we measured IL-18-induced IFN-{gamma} production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-{gamma} expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-{gamma} production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-{gamma} production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-{gamma} production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-{gamma} production via p38 MAPK.

  8. MAPKs in development: insights from Dictyostelium signaling pathways

    PubMed Central

    Hadwiger, Jeffrey A.; Nguyen, Hoai-Nghia

    2011-01-01

    Mitogen activated protein kinases (MAPKs) play important roles in the development of eukaryotic organisms through the regulation of signal transduction pathways stimulated by external signals. MAPK signaling pathways have been associated with the regulation of cell growth, differentiation, and chemotaxis, indicating MAPKs contribute to a diverse set of developmental processes. In most eukaryotes, the diversity of external signals is likely to far exceed the diversity of MAPKs, suggesting that multiple signaling pathways might share MAPKs. Do different signaling pathways converge before MAPK function or can MAPKs maintain signaling specificity through interactions with specific proteins? The genetic and biochemical analysis of MAPK pathways in simple eukaryotes such as Dictyostelium offers opportunities to investigate functional specificity of MAPKs in G protein-mediated signal transduction pathways. This review considers the regulation and specificity of MAPK function in pathways that control Dictyostelium growth and development. PMID:21666837

  9. FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct.

    PubMed

    Terakawa, Jumpei; Rocchi, Altea; Serna, Vanida A; Bottinger, Erwin P; Graff, Jonathan M; Kurita, Takeshi

    2016-07-01

    Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate. PMID:27164167

  10. Roles for the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, in feedback control of inflammatory gene expression and repression by dexamethasone.

    PubMed

    Shah, Suharsh; King, Elizabeth M; Chandrasekhar, Ambika; Newton, Robert

    2014-05-01

    Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression. PMID:24692548

  11. Exocyst Sec10 protects epithelial barrier integrity and enhances recovery following oxidative stress, by activation of the MAPK pathway.

    PubMed

    Park, Kwon Moo; Fogelgren, Ben; Zuo, Xiaofeng; Kim, Jinu; Chung, Daniel C; Lipschutz, Joshua H

    2010-03-01

    Cell-cell contacts are essential for epithelial cell function, and disruption is associated with pathological conditions including ischemic kidney injury. We hypothesize that the exocyst, a highly-conserved eight-protein complex that targets secretory vesicles carrying membrane proteins, is involved in maintaining renal epithelial barrier integrity. Accordingly, increasing exocyst expression in renal tubule cells may protect barrier function from oxidative stress resulting from ischemia and reperfusion (I/R) injury. When cultured on plastic, Madin-Darby canine kidney (MDCK) cells overexpressing Sec10, a central exocyst component, formed domes showing increased resistance to hydrogen peroxide (H2O2). Transepithelial electric resistance (TER) of Sec10-overexpressing MDCK cells grown on Transwell filters was higher than in control MDCK cells, and the rate of TER decrease following H2O2 treatment was less in Sec10-overexpressing MDCK cells compared with control MDCK cells. After removal of H2O2, TER returned to normal more rapidly in Sec10-overexpressing compared with control MDCK cells. In collagen culture MDCK cells form cysts, and H2O2 treatment damaged Sec10-overexpressing MDCK cell cysts less than control MDCK cell cysts. The MAPK pathway has been shown to protect animals from I/R injury. Levels of active ERK, the final MAPK pathway step, were higher in Sec10-overexpressing compared with control MDCK cells. U0126 inhibited ERK activation, exacerbated the H2O2-induced decrease in TER and cyst disruption, and delayed recovery of TER following H2O2 removal. Finally, in mice with renal I/R injury, exocyst expression decreased early and returned to normal concomitant with functional recovery, suggesting that the exocyst may be involved in the recovery following I/R injury.

  12. ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells.

    PubMed

    Banerjee, Sayantan; McGee, Dennis W

    2016-09-01

    Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation.

  13. Deficiency of MAPK-activated protein kinase 2 (MK2) prevents adverse remodelling and promotes endothelial healing after arterial injury.

    PubMed

    Kapopara, P R; von Felden, J; Soehnlein, O; Wang, Y; Napp, L C; Sonnenschein, K; Wollert, K C; Schieffer, B; Gaestel, M; Bauersachs, J; Bavendiek, U

    2014-12-01

    Maladaptive remodelling of the arterial wall after mechanical injury (e. g. angioplasty) is characterised by inflammation, neointima formation and media hypertrophy, resulting in narrowing of the affected artery. Moreover, mechanical injury of the arterial wall causes loss of the vessel protecting endothelial cell monolayer. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a major downstream target of p38 MAPK, regulates inflammation, cell migration and proliferation, essential processes for vascular remodelling and re-endothelialisation. Therefore, we investigated the role of MK2 in remodelling and re-endothelialisation after arterial injury in genetically modified mice in vivo. Hypercholesterolaemic low-density-lipoprotein-receptor-deficient mice (ldlr-/-) were subjected to wire injury of the common carotid artery. MK2-deficiency (ldlr-/-/mk2-/-) nearly completely prevented neointima formation, media hypertrophy, and lumen loss after injury. This was accompanied by reduced proliferation and migration of MK2-deficient smooth muscle cells. In addition, MK2-deficiency severely reduced monocyte adhesion to the arterial wall (day 3 after injury, intravital microscopy), which may be attributed to reduced expression of the chemokine ligands CCL2 and CCL5. In line, MK2-deficiency significantly reduced the content of monocytes, neutrophiles and lymphocytes of the arterial wall (day 7 after injury, flow cytometry). In conclusion, in a model of endothelial injury (electric injury), MK2-deficiency strongly increased proliferation of endothelial cells and improved re-endothelialisation of the arterial wall after injury. Deficiency of MK2 prevents adverse remodelling and promotes endothelial healing of the arterial wall after injury, suggesting that MK2-inhibition is a very attractive intervention to prevent restenosis after percutaneous therapeutic angioplasty. PMID:25120198

  14. Thrombosis recanalization by paeoniflorin through the upregulation of urokinase‑type plasminogen activator via the MAPK signaling pathway.

    PubMed

    Ye, Songshan; Mao, Bingyu; Yang, Lei; Fu, Weiyun; Hou, Junran

    2016-06-01

    Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen‑activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme‑linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6‑keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D‑dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti‑thrombosis pathways. PMID:27082639

  15. ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells.

    PubMed

    Banerjee, Sayantan; McGee, Dennis W

    2016-09-01

    Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation. PMID:27173611

  16. Role of p38 MAPK activation and mitochondrial cytochrome-c release in allicin-induced apoptosis in SK-N-SH cells.

    PubMed

    Zhuang, Jianhui; Li, Yu; Chi, Yufen

    2016-04-01

    Here, we investigate the apoptotic effect of allicin, the predominant component of freshly crushed garlic, on neuroblastoma cells. In this paper, the authors have first assessed the effect of allicin on human neuroblastoma SK-N-SH cells and then investigated the underlying mechanism. The results indicate that allicin suppresses SK-N-SH cell growth in a dose-dependent and time-dependent manner and that 5 μmol/l of allicin leads to a significant increase in apoptotic rate with annexin-V/PI double staining. Western blot analysis shows that treatment with allicin-induced apoptosis through activation of caspases-3 and 9. Phosphorylation of p38 MAPK contributes to allicin-induced apoptosis upstream of caspase activation. Using p38 MAPK inhibitor, the authors discovered that p38 MAPK activation subsequently induces the release of cytochrome-c from mitochondria into the cytosol. Taken together, the results demonstrate that allicin can activate the p38 MAPK pathway, which leads to mitochondrial release of cytochrome-c, thus inducing SK-N-SH cell apoptosis. Overall, this study suggests that allicin may be used as one of the novel pharmacological treatment strategies in neuroblastoma. PMID:26771864

  17. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells.

    PubMed

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying; Zhao, Jie

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism. PMID:27635402

  18. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    PubMed Central

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  19. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    PubMed Central

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism. PMID:27635402

  20. A novel mechanism for momordin Ic-induced HepG2 apoptosis: involvement of PI3K- and MAPK-dependent PPARγ activation.

    PubMed

    Wang, Jing; Yuan, Li; Xiao, Haifang; Wang, Chan; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2014-05-01

    Momordin Ic is a natural triterpenoid saponin found in various Chinese and Japanese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. Momordin Ic has been previously demonstrated to induce HepG2 cell apoptosis in a ROS-mediated PI3K and MAPK pathway-dependent manner. In the present study, the underlying mechanisms of PI3K and MAPK pathway-mediated PPARγ, and PGC-1α co-regulator activation, as well as the effects of downstream proteins, COX-2 and FoxO4, on cell apoptosis were investigated. The results demonstrated that momordin Ic activated PPARγ and inhibited COX-2. PGC-1α and FoxO4 expressions were increased by the PI3K or MAPK pathways. Furthermore, PPARγ inhibition decreased p-p38 and FoxO4 expression, and restored COX-2 expression. ROS inhibition exerted little effect on PPARγ, COX-2 and FoxO4 expression but affected PGC-1α expression. These results revealed the involvement of PI3K and MAPK-dependent PPARγ activation in momordin Ic-induced apoptosis, providing more detailed information underlying the pro-apoptotic mechanism of momordin Ic in HepG2 cell apoptosis. PMID:24584198

  1. Stimulation of Ebola virus production from persistent infection through activation of the Ras/MAPK pathway.

    PubMed

    Strong, James E; Wong, Gary; Jones, Shane E; Grolla, Allen; Theriault, Steven; Kobinger, Gary P; Feldmann, Heinz

    2008-11-18

    Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV "jumps" from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2alpha phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo. Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the "hit-and-run" nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission. PMID:18981410

  2. The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence

    PubMed Central

    Thompson, Keyata N.; Whipple, Rebecca A.; Yoon, Jennifer R.; Lipsky, Michael; Charpentier, Monica S.; Boggs, Amanda E.; Chakrabarti, Kristi R.; Bhandary, Lekhana; Hessler, Lindsay K.; Martin, Stuart S.; Vitolo, Michele I.

    2015-01-01

    A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN−/−KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common. PMID:26497685

  3. The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence.

    PubMed

    Thompson, Keyata N; Whipple, Rebecca A; Yoon, Jennifer R; Lipsky, Michael; Charpentier, Monica S; Boggs, Amanda E; Chakrabarti, Kristi R; Bhandary, Lekhana; Hessler, Lindsay K; Martin, Stuart S; Vitolo, Michele I

    2015-11-01

    A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common. PMID:26497685

  4. Inhibition of inflammatory mediators contributes to the anti-inflammatory activity of KYKZL-1 via MAPK and NF-κB pathway

    SciTech Connect

    Xu, Guang-Lin; Du, Yi-Fang; Cheng, Jing; Huan, Lin; Chen, Shi-Cui; Wei, Shao-Hua; Gong, Zhu-Nan; Cai, Jie; Qiu, Ting; Wu, Hao; Sun, Ting; Ao, Gui-Zhen

    2013-10-01

    KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the anti-inflammatory activity test focusing on its modulation of inflammatory mediators as well as intracellular MAPK and NF-κB signaling pathways. In acute ear edema model, pretreatment with KYKZL-1 (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice with a higher inhibition than diclofenac. In a three-day TPA-induced inflammation, KYKZL-1 also showed significant anti-inflammatory activity with inhibition ranging between 20% and 64%. In gastric lesion test, KYKZL-1 elicited markedly fewer stomach lesions with a low index of ulcer as compared to diclofenac in rats. In further studies, KYKZL-1 was found to significantly inhibit the production of NO, PGE{sub 2}, LTB{sub 4} in LPS challenged RAW264.7, which is parallel to its attenuation of the expression of iNOS, COX-2, 5-LOX mRNAs or proteins and inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. Taken together, our data indicate that KYKZL-1 comprises dual inhibition of COX and 5-LOX and exerts an obvious anti-inflammatory activity with an enhanced gastric safety profile via simultaneous inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. - Highlights: • KYKZL-1 is designed to exhibit COX/5-LOX dual inhibition. • KYKZL-1 inhibits NO, PGE{sub 2} and LTB{sub 4} and iNOS, COX-2 and 5-LOX mRNAs and MAPKs. • KYKZL-1 inhibits phosphorylation of MAPKs. • KYKZL-1 inactivates NF-κB pathway.

  5. Carbon monoxide alleviates ethanol-induced oxidative damage and inflammatory stress through activating p38 MAPK pathway

    SciTech Connect

    Li, Yanyan; Gao, Chao; Shi, Yanru; Tang, Yuhan; Liu, Liang; Xiong, Ting; Du, Min; Xing, Mingyou; Liu, Liegang; Yao, Ping

    2013-11-15

    Stress-inducible protein heme oxygenase-1(HO-1) is well-appreciative to counteract oxidative damage and inflammatory stress involving the pathogenesis of alcoholic liver diseases (ALD). The potential role and signaling pathways of HO-1 metabolite carbon monoxide (CO), however, still remained unclear. To explore the precise mechanisms, ethanol-dosed adult male Balb/c mice (5.0 g/kg.bw.) or ethanol-incubated primary rat hepatocytes (100 mmol/L) were pretreated by tricarbonyldichlororuthenium (II) dimmer (CORM-2, 8 mg/kg for mice or 20 μmol/L for hepatocytes), as well as other pharmacological reagents. Our data showed that CO released from HO-1 induction by quercetin prevented ethanol-derived oxidative injury, which was abolished by CO scavenger hemoglobin. The protection was mimicked by CORM-2 with the attenuation of GSH depletion, SOD inactivation, MDA overproduction, and the leakage of AST, ALT or LDH in serum and culture medium induced by ethanol. Moreover, CORM-2 injection or incubation stimulated p38 phosphorylation and suppressed abnormal Tnfa and IL-6, accompanying the alleviation of redox imbalance induced by ethanol and aggravated by inflammatory factors. The protective role of CORM-2 was abolished by SB203580 (p38 inhibitor) but not by PD98059 (ERK inhibitor) or SP600125 (JNK inhibitor). Thus, HO-1 released CO prevented ethanol-elicited hepatic oxidative damage and inflammatory stress through activating p38 MAPK pathway, suggesting a potential therapeutic role of gaseous signal molecule on ALD induced by naturally occurring phytochemicals. - Highlights: • CO alleviated ethanol-derived liver oxidative and inflammatory stress in mice. • CO eased ethanol and inflammatory factor-induced oxidative damage in hepatocytes. • The p38 MAPK is a key signaling mechanism for the protective function of CO in ALD.

  6. A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC

    PubMed Central

    Regazzo, Daniela; Bertazza, Loris; Galuppini, Francesca; Guzzardo, Vincenza; Jaffrain-Rea, Marie Lise; Vianello, Federica; Ciato, Denis; Ceccato, Filippo; Watutantrige-Fernando, Sara; Bisognin, Andrea; Bortoluzzi, Stefania; Pennelli, Gianmaria; Boscaro, Marco; Scaroni, Carla; Mian, Caterina

    2015-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAFV600E may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice. PMID:26392334

  7. Lipopolysaccharide enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

    PubMed Central

    Luo, Shue-Fen; Wang, Chuan-Chwan; Chiu, Chi-Tso; Chien, Chin-Sung; Hsiao, Li-Der; Lin, Chien-Huang; Yang, Chuen-Mao

    2000-01-01

    Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen-activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)-induced inositol phosphates (IPs) accumulation and Ca2+ mobilization in canine cultured tracheal smooth muscle cells (TSMCs).LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentration-dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achieved with 100 μg ml−1 LPS.Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation and Ca2+ mobilization. However, there was no effect on the IPs response induced by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF-BB enhanced BK-induced IPs response.These enhancements by LPS and PDGF-BB might be due to an increase in BK B2 receptor density (Bmax) in TSMCs, characterized by competitive inhibition of [3H]-BK binding using B1 and B2 receptor-selective reagents.The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs.Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases.These results suggest that the augmentation of BK-induced responses produced by LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs. PMID:10952668

  8. RNase activity of sialic acid-binding lectin from bullfrog eggs drives antitumor effect via the activation of p38 MAPK to caspase-3/7 signaling pathway in human breast cancer cells

    PubMed Central

    Kariya, Yukiko; Tatsuta, Takeo; Sugawara, Shigeki; Kariya, Yoshinobu; Nitta, Kazuo; Hosono, Masahiro

    2016-01-01

    Sialic acid-binding lectin obtained from bullfrog eggs (SBL) induces cell death in cancer cells but not in normal cells. This antitumor effect is mediated through its ribo-nuclease (RNase) activity. However, the underlying molecular mechanisms remain unclear. We found that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was activated when SBL induced cell death in three human breast cancer cell lines: SK-BR-3, MCF-7, and MDA-MB231. The suppression of p38 MAPK phosphorylation by a p38 MAPK inhibitor as well as short interference RNA knockdown of p38 MAPK expression significantly decreased cell death and increased the cell viability of SBL-treated MDA-MB231 cells. H103A, an SBL mutant lacking in RNase activity, showed decreased SBL-induced cell death compared with native SBL. However, the loss of RNase activity of SBL had no effect on its internalization into cells. The H103A mutant also displayed decreased phosphorylation of p38 MAPK. Moreover, SBL promoted caspase-3/7 activation followed by a cleavage of poly (ADP-ribose)-polymerase, whereas the SBL mutant, H103A, lost this ability. The SBL-induced caspase-3/7 activation was suppressed by the p38 MAPK inhibitor, SB203580, as well as pan-caspase inhibitor, zVAD-fmk. In the presence of zVAD-fmk, the SBL-induced cell death was decreased. In addition, the cell viability of SBL-treated MDA-MB231 cells recovered by zVAD-fmk treatment. Taken together, our results suggest that the RNase activity of SBL leads to breast cancer cell death through the activation of p38 MAPK followed by the activation of caspase-3/7. PMID:27513956

  9. A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways

    PubMed Central

    Kim, Byung-Wook; More, Sandeep Vasant; Yun, Yo-Sep; Ko, Hyun-Myung; Kwak, Jae-Hwan; Lee, Heesoon; Suk, Kyoungho; Kim, In-Su; Choi, Dong-Kug

    2016-01-01

    Aim: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro. Methods: Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence microscopy, respectively. Results: Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). Conclusion: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells. PMID:26838070

  10. HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation

    PubMed Central

    Muthumani, Karuppiah; Choo, Andrew Y.; Hwang, Daniel S.; Premkumar, Arumugam; Dayes, Nathanael S.; Harris, Crafford; Green, Douglas R.; Wadsworth, Scott A.; Siekierka, John J.; Weiner, David B.

    2005-01-01

    The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro. (Blood. 2005;106:2059-2068) PMID:15928037

  11. Role of P38 MAPK on MMP Activity in Photothrombotic Stroke Mice as Measured using an Ultrafast MMP Activatable Probe

    PubMed Central

    Chang, Di; Wang, Yuan-Cheng; Bai, Ying-Ying; Lu, Chun-Qiang; Xu, Ting-Ting; Zhu, Lei; Ju, Shenghong

    2015-01-01

    Matrix metalloproteinases (MMPs) exert a dual effect in ischemic stroke and thus represent an ideal target for detection and therapy. However, to date, all clinical trials of MMP inhibitors have failed, and alternative drug candidates and therapeutic targets are urgently required. Nonetheless, further investigations are limited by the lack of non-invasive imaging techniques. Here, we report a novel, fast and ultrasensitive MMP activatable optical imaging probe for the dynamic visualization of MMP activity in photothrombotic stroke mice. This probe provides a significant signal enhancement in as little as 15 min, with the highest signal intensity occurring at 1 h post-injection, and shows high sensitivity in measuring MMP activity alterations, which makes it specifically suitable for the real-time visualization of MMP activity and drug discovery in preclinical research. Moreover, using this probe, we successfully demonstrate that the regulation of the p38 mitogen-activated protein kinase (MAPK) signal pathway is capable of modulating MMP activity after stroke, revealing a novel regulatory mechanism of postischemic brain damage and overcoming the limitations of traditional therapeutic strategies associated with MMP inhibitors by using a non-invasive molecular imaging method. PMID:26581247

  12. Upregulation of proinflammatory cytokines and NO production in BV-activated avian macrophage-like cell line (HD11) requires MAPK and NF-kappaB pathways.

    PubMed

    Han, Yan; Niu, Mingshan; An, Lijia; Li, Wenli

    2009-07-01

    The budded virus (BV) of Antheraea pernyi is a nuclear polyhedrosis virus (ApNPV), that belongs to the baculovirus family. It is capable of stimulating innate immune response in avian macrophage-like cells (HD11). In this study, we used the expression of proinflammatory cytokines (IFN-gamma and IL-12p40) and production of nitric oxide (NO) as indicators to evaluate the involvement of the signaling cascades in the activation of HD11 cells by BV. Western blot analysis revealed that ERK1/2, p38 MAPK and JNK were phosphorylated followed by activation of p65-NF-kappaB after HD11 cells were challenged with BV. Inhibition of p38 MAPK and NF-kappaB by their respective inhibitors abolished BV-induced expression of IL-12p40, IFN-gamma and production of NO, whereas inhibition of JNK abolished only the induction of cytokines. Additionally, inhibition of ERK1/2 resulted in unexpected increases in both cytokine expression and NO production. Taken together, these findings indicated that in BV-induced HD11 cells, the induction of cytokines was mediated by the p38 MAPK, JNK and NF-kappaB pathways, and NO production was mediated through the p38 MAPK/NF-kappaB pathways.

  13. Hyaluronan Oligosaccharides Induce MMP-1 and -3 via Transcriptional Activation of NF-κB and p38 MAPK in Rheumatoid Synovial Fibroblasts

    PubMed Central

    Hanabayashi, Masahiro; Takahashi, Nobunori; Sobue, Yasumori; Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    Objective To explore the effect of hyaluronan oligosaccharides (HAoligos) on interactions between HA and its principal receptor, CD44, in rheumatoid synovial fibroblasts (RSFs) and matrix metalloproteinase (MMP) production. Methods RSFs were isolated from rheumatoid synovial tissue. HA distribution was visualized by immunocytochemistry. MMP-1 and MMP-3 induction was analyzed by real-time RT-PCR and immunoblotting. The interaction between HAoligos and their MMP-producing receptors was tested by blocking with anti-CD44 and anti-Toll-like receptor 4 (TLR-4). Phosphorylation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting. Results Endogenous HA decreased after treatment with HAoligos, while MMP-1 and MMP-3 expression increased in a dose-dependent manner. Pretreatment with anti-CD44 or anti-TLR-4 antibody significantly reduced the effect of HAoligos on MMP-1 and MMP-3 mRNA expression. NF-κB and p38 MAPK phosphorylation was enhanced by HAoligos pretreated with anti-TLR-4, and HAoligo-induced MMP production was blocked with an inhibitor of NF-κB and p38 MAPK pathways. Conclusions Disruptive changes in CD44-HA interactions by HAoligos enhanced MMP-1 and MMP-3 production via activation of NF-κB and p38 MAPK signaling pathways in RSFs. PMID:27564851

  14. Mammary gland specific expression of Brk/PTK6 promotes delayed involution and tumor formation associated with activation of p38 MAPK

    PubMed Central

    2011-01-01

    Introduction Protein tyrosine kinases (PTKs) are frequently overexpressed and/or activated in human malignancies, and regulate cancer cell proliferation, cellular survival, and migration. As such, they have become promising molecular targets for new therapies. The non-receptor PTK termed breast tumor kinase (Brk/PTK6) is overexpressed in approximately 86% of human breast tumors. The role of Brk in breast pathology is unclear. Methods We expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed mammary glands from wild-type (wt) and transgenic mice after forced weaning. Western blotting and immunohistochemistry (IHC) studies were conducted to visualize markers of mammary gland involution, cell proliferation and apoptosis, as well as Brk, STAT3, and activated p38 mitogen-activated protein kinase (MAPK) in mammary tissues and tumors from WAP-Brk mice. Human (HMEC) or mouse (HC11) mammary epithelial cells were stably or transiently transfected with Brk cDNA to assay p38 MAPK signaling and cell survival in suspension or in response to chemotherapeutic agents. Results Brk-transgenic dams exhibited delayed mammary gland involution and aged mice developed infrequent tumors with reduced latency relative to wt mice. Consistent with delayed involution, mammary glands of transgenic animals displayed decreased STAT3 phosphorylation, a marker of early-stage involution. Notably, p38 MAPK, a pro-survival signaling mediator downstream of Brk, was activated in mammary glands of Brk transgenic relative to wt mice. Brk-dependent signaling to p38 MAPK was recapitulated by Brk overexpression in the HC11 murine mammary epithelial cell (MEC) line and human MEC, while Brk knock-down in breast cancer cells blocked EGF-stimulated p38 signaling. Additionally, human or mouse MECs expressing Brk exhibited increased anchorage-independent survival and resistance to doxorubicin. Finally, breast tumor biopsies were subjected to IHC analysis for co-expression of Brk and phospho-p38 MAPK

  15. 6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA.

    PubMed

    Gomez-Lazaro, Maria; Galindo, Maria F; Concannon, Caoimhín G; Segura, Miguel F; Fernandez-Gomez, Francisco J; Llecha, Nuria; Comella, Joan X; Prehn, Jochen H M; Jordan, Joaquin

    2008-03-01

    Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.

  16. Selective Activation of Human Dendritic Cells by OM-85 through a NF-kB and MAPK Dependent Pathway

    PubMed Central

    Scutera, Sara; Somma, Paolo; Salvi, Valentina; Musso, Tiziana; Tabbia, Giuseppe; Bardessono, Marco; Pasquali, Christian; Mantovani, Alberto; Sozzani, Silvano; Bosisio, Daniela

    2013-01-01

    OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings. PMID:24386121

  17. Molecular cloning of the duck mitogen-activated protein kinase 1 (MAPK1) gene and the development of a quantitative real-time PCR assay to detect its expression.

    PubMed

    Cao, Shoulin; Han, Xiangan; Ding, Chan; Wang, Shaohui; Tian, Mingxing; Wang, Xiaolan; Hou, Wanwan; Yue, Jiaping; Wang, Guijun; Yu, Shengqing

    2014-09-01

    Mitogen-activated protein kinase 1 (MAPK1) acts as an integration point for multiple biochemical signals, and is involved in a wide variety of biological processes such as cell proliferation and differentiation, transcription regulation, and development. Mitogen-activated protein kinase 1 plays an important role in inducing cell death in bacterial infections. In this study, the duck MAPK1 gene was cloned for the first time from the Cherry Valley duck. Sequence analysis showed that duck MAPK1 cDNA is 1,557 bp long, with an open reading frame of 1,107 bp. It encodes 368 amino acids, with 85.4, 84.5, and 97.3% homology with the human, mouse, and chicken MAPK1 gene, respectively. Furthermore, a SYBR Green quantitative real-time PCR assay was developed to detect duck MAPK1 expression. Following Riemerella anatipestifer infection by virulent strain Yb2, MAPK1 mRNA level increased more than 200-fold in the duck spleens, suggesting that increased duck MAPK1 expression can be used as an indicator of bacterial infection. Our results provide ground work to warrant further studies of the duck MAPK1 gene in bacterial pathogenesis.

  18. Molecular cloning of the duck mitogen-activated protein kinase 1 (MAPK1) gene and the development of a quantitative real-time PCR assay to detect its expression.

    PubMed

    Cao, Shoulin; Han, Xiangan; Ding, Chan; Wang, Shaohui; Tian, Mingxing; Wang, Xiaolan; Hou, Wanwan; Yue, Jiaping; Wang, Guijun; Yu, Shengqing

    2014-09-01

    Mitogen-activated protein kinase 1 (MAPK1) acts as an integration point for multiple biochemical signals, and is involved in a wide variety of biological processes such as cell proliferation and differentiation, transcription regulation, and development. Mitogen-activated protein kinase 1 plays an important role in inducing cell death in bacterial infections. In this study, the duck MAPK1 gene was cloned for the first time from the Cherry Valley duck. Sequence analysis showed that duck MAPK1 cDNA is 1,557 bp long, with an open reading frame of 1,107 bp. It encodes 368 amino acids, with 85.4, 84.5, and 97.3% homology with the human, mouse, and chicken MAPK1 gene, respectively. Furthermore, a SYBR Green quantitative real-time PCR assay was developed to detect duck MAPK1 expression. Following Riemerella anatipestifer infection by virulent strain Yb2, MAPK1 mRNA level increased more than 200-fold in the duck spleens, suggesting that increased duck MAPK1 expression can be used as an indicator of bacterial infection. Our results provide ground work to warrant further studies of the duck MAPK1 gene in bacterial pathogenesis. PMID:24974389

  19. PPAR{alpha} agonist fenofibrate protects the kidney from hypertensive injury in spontaneously hypertensive rats via inhibition of oxidative stress and MAPK activity

    SciTech Connect

    Hou, Xiaoyang; Shen, Ying H.; Li, Chuanbao; Wang, Fei; Zhang, Cheng; Bu, Peili; Zhang, Yun

    2010-04-09

    Oxidative stress has been shown to play an important role in the development of hypertensive renal injury. Peroxisome proliferator-activated receptors {alpha} (PPAR{alpha}) has antioxidant effect. In this study, we demonstrated that fenofibrate significantly reduced proteinuria, inflammatory cell recruitment and extracellular matrix (ECM) proteins deposition in the kidney of SHRs without apparent effect on blood pressure. To investigate the mechanisms involved, we found that fenofibrate treatment markedly reduced oxidative stress accompanied by reduced activity of renal NAD(P)H oxidase, increased activity of Cu/Zn SOD, and decreased phosphorylation of p38MAPK and JNK in the kidney of SHRs. Taken together, fenofibrate treatment can protect against hypertensive renal injury without affecting blood pressure by inhibiting inflammation and fibrosis via suppression of oxidative stress and MAPK activity.

  20. Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia.

    PubMed

    Funk, Adam J; McCullumsmith, Robert E; Haroutunian, Vahram; Meador-Woodruff, James H

    2012-03-01

    Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. PMID:22048463

  1. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity

    PubMed Central

    Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  2. Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

    PubMed

    Pich, Christine; Teiti, Iotefa; Sarrabayrouse, Guillaume; Gallardo, Franck; Gence, Rémi; Tilkin-Mariamé, Anne-Françoise

    2016-01-01

    CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors. PMID:26828592

  3. Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells.

    PubMed

    Zhu, Liqian; Ding, Xiuyan; Zhu, Xiaofang; Meng, Songshu; Wang, Jianye; Zhou, Hong; Duan, Qiangde; Tao, Jie; Schifferli, Dieter M; Zhu, Guoqiang

    2011-04-14

    Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1) infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.

  4. Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism.

    PubMed

    Axmann, A; Seidel, D; Reimann, T; Hempel, U; Wenzel, K W

    1998-08-19

    In fibroblasts transforming growth factor-beta1 (TGF-beta1) regulates cell proliferation and turnover of macromolecular components of the extracellular matrix. Here, intracellular signaling events in growth-inhibited embryonic rat lung fibroblasts (RFL-6) upon stimulation with TGF-beta1 were investigated. TGF-beta1 rapidly induced the activation of c-Raf-1, MEK-1, and MAPK p42 and p44. The activation of this pathway by TGF-beta1 did not depend on autocrine platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). Inhibition of the binding of growth factors to their tyrosine kinase receptors did not affect MAPK activation by TGF-beta1. Ras activation by TGF-beta1 was significantly lower compared to the activation by PDGF or bFGF. The intracellular transduction of the TGF-beta1 signal was completely suppressed by depletion or inhibition of protein kinase C (PKC). It is shown that calcium-dependent isoforms of PKC are required for MAPK activation by TGF-beta1. PMID:9712718

  5. Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK.

    PubMed

    Chang, Jia; Sonoyama, Wataru; Wang, Zhuo; Jin, Qiming; Zhang, Chengfei; Krebsbach, Paul H; Giannobile, William; Shi, Songtao; Wang, Cun-Yu

    2007-10-19

    Mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into osteoblasts and provide an excellent cell source for bone regeneration and repair. Recently, the canonical Wnt/beta-catenin signaling pathway has been found to play a critical role in skeletal development and osteogenesis, implying that Wnts can be utilized to improve de novo bone formation mediated by MSCs. However, it is unknown whether noncanonical Wnt signaling regulates osteogenic differentiation. Here, we find that Wnt-4 enhanced in vitro osteogenic differentiation of MSCs isolated from human adult craniofacial tissues and promoted bone formation in vivo. Whereas Wnt-4 did not stabilize beta-catenin, it activated p38 MAPK in a novel noncanonical signaling pathway. The activation of p38 was dependent on Axin and was required for the enhancement of MSC differentiation by Wnt-4. Moreover, using two different models of craniofacial bone injury, we found that MSCs genetically engineered to express Wnt-4 enhanced osteogenesis and improved the repair of craniofacial defects in vivo. Taken together, our results reveal that noncanonical Wnt signaling could also play a role in osteogenic differentiation. Wnt-4 may have a potential use in improving bone regeneration and repair of craniofacial defects.

  6. Neferine, an alkaloid from lotus seed embryo, inhibits human lung cancer cell growth by MAPK activation and cell cycle arrest.

    PubMed

    Poornima, Paramasivan; Weng, Ching Feng; Padma, Viswanadha Vijaya

    2014-01-01

    Neferine is the major bisbenzylisoquinoline alkaloid isolated from the seed embryo of a traditional medicinal plant Nelumbo nucifera (Lotus). Epidemiological studies have revealed the therapeutic potential of lotus seed embryo. Although several mechanisms have been proposed, a clear anticancer action mechanism of neferine on lung cancer cells is still not known. Lung cancer is the most common cause of cancer death in the world, and the patients with advanced stage of nonsmall lung cancer require adjunct chemotherapy after surgical resection for the eradication of cancer cells. In this study, the effects of neferine were evaluated and characterized in A549 cells. Neferine induced apoptosis in a dose-dependent manner with the hypergeneration of reactive oxygen species, activation of MAPKs, lipid peroxidation, depletion of cellular antioxidant pool, loss of mitochondrial membrane potential, and intracellular calcium accumulation. Furthermore, neferine treatment leads to the inhibition of nuclear factor kappaB and Bcl2, upregulation of Bax and Bad, release of cytochrome C, activation of caspase cascade, and DNA fragmentation. In addition, neferine could induce p53 and its effector protein p21 and downregulation of cell cycle regulatory protein cyclin D1 thereby inducing G1 cell cycle arrest. These results suggest a novel function of neferine as an apoptosis inducer in lung cancer cells.

  7. Iron induces hepatocytes death via MAPK activation and mitochondria-dependent apoptotic pathway: beneficial role of glycine.

    PubMed

    Bhattacharyya, Sudip; Ghosh, Jyotirmoy; Sil, Parames C

    2012-10-01

    In the present study we investigated the beneficial role of glycine in iron (FeSO₄) induced oxidative damage in murine hepatocytes. Exposure of hepatocytes to 20 μM FeSO₄ for 3 hours enhanced reactive oxygen species (ROS) generation and induced alteration in biochemical parameters related to hepatic oxidative stress. Investigating cell signalling pathway, we observed that iron (FeSO₄) intoxication caused NF-κB activation as well as the phosphorylation of p38 and ERK MAPKs. Iron (FeSO₄) administration also disrupted Bcl-2/Bad protein balance, reduced mitochondrial membrane potential, released cytochrome c and induced the activation of caspases and cleavage of PARP protein. Flow cytometric analysis also confirmed that iron (FeSO₄) induced hepatocytes death is apoptotic in nature. Glycine (10 mM) supplementation, on the other hand, reduced all the iron (FeSO₄) induced apoptotic indices. Combining, results suggest that glycine could be a beneficial agent against iron mediated toxicity in hepatocytes. PMID:22817335

  8. Activation of transient receptor potential vanilloid 4 induces apoptosis in hippocampus through downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways

    PubMed Central

    Jie, P; Hong, Z; Tian, Y; Li, Y; Lin, L; Zhou, L; Du, Y; Chen, L; Chen, L

    2015-01-01

    Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that is sensitive to cell swelling, arachidonic acid and its metabolites, epoxyeicosatrienoic acids, which are associated with cerebral ischemia. The activation of TRPV4 induces cytotoxicity in many types of cells, accompanied by an increase in the intracellular free calcium concentration. TRPV4 activation modulates the mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K)/ protein kinase B (Akt) signaling pathways that regulate cell death and survival. Herein, we examined TRPV4-induced neuronal apoptosis by intracerebroventricular (ICV) injection of a TRPV4 agonist (GSK1016790A) and assessed its involvement in cerebral ischemic injury. ICV injection of GSK1016790A dose-dependently induced apoptosis in the mouse hippocampi (GSK-injected mice). The protein level of phosphorylated p38 MAPK (p-p38 MAPK) was markedly increased and that of phosphorylated c-Jun N-terminal protein kinase (p-JNK) was virtually unchanged. TRPV4 activation also decreased Bcl-2/Bax protein ratio and increased the cleaved caspase-3 protein level, and these effects were blocked by a PI3K agonist and a p38 MAPK antagonist, but were unaffected by a JNK antagonist. ICV injection of the TRPV4 antagonist HC-067047 reduced brain infarction after reperfusion for 48 h in mice with middle cerebral artery occlusion (MCAO). In addition, HC-067047 treatment attenuated the decrease in the phosphorylated Akt protein level and the increase in p-p38 MAPK protein level at 48 h after MCAO, while the increase in p-JNK protein level remained unchanged. Finally, the decreased Bcl-2/Bax protein ratio and the increased cleaved caspase-3 protein level at 48 h after MCAO were markedly attenuated by HC-067047. We conclude that activation of TRPV4 induces apoptosis by downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways, which is involved in cerebral ischemic injury. PMID:26043075

  9. Attenuation of the macrophage inflammatory activity by TiO₂ nanotubes via inhibition of MAPK and NF-κB pathways.

    PubMed

    Neacsu, Patricia; Mazare, Anca; Schmuki, Patrik; Cimpean, Anisoara

    2015-01-01

    Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity.

  10. Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes.

    PubMed

    Schuon, C; Ebeling, S; Meinecke, B

    2007-08-01

    The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus-oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600-800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22-24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.

  11. γ-Enolase C-terminal peptide promotes cell survival and neurite outgrowth by activation of the PI3K/Akt and MAPK/ERK signalling pathways.

    PubMed

    Hafner, Anja; Obermajer, Nataša; Kos, Janko

    2012-04-15

    γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. In the present study, we investigated the involvement of γ-enolase in PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling, the two pathways triggered predominantly by neurotrophic factors. Whereas the PI3K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI3K and ERK1/2, leading to subsequent expression of the growth-cone-specific protein GAP-43 (growth-associated protein of 43 kDa). MEK (MAPK/ERK kinase) and PI3K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodelling of the actin-based cytoskeleton. We show that γ-enolase-mediated PI3K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA downstream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase-induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration by activating the PI3K/Akt and MAPK/ERK signalling pathways, resulting in downstream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodelling, activation of transcriptional factors and regulation of the cell cycle.

  12. Attenuation of the macrophage inflammatory activity by TiO2 nanotubes via inhibition of MAPK and NF-κB pathways

    PubMed Central

    Neacsu, Patricia; Mazare, Anca; Schmuki, Patrik; Cimpean, Anisoara

    2015-01-01

    Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity. PMID:26491301

  13. Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation

    PubMed Central

    Endale, M; Lee, WM; Kamruzzaman, SM; Kim, SD; Park, JY; Park, MH; Park, TY; Park, HJ; Cho, JY; Rhee, MH

    2012-01-01

    BACKGROUND AND PURPOSE Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti-platelet activities have shown stability and bioavailability limitations. However, information on the anti-platelet activity of ginsenoside-Rp1 (G-Rp1), a stable derivative of ginsenoside-Rg3, is scarce. We examined the ability of G-Rp1 to modulate agonist-induced platelet activation. EXPERIMENTAL APPROACH G-Rp1 in vitro and ex vivo effects on agonist-induced platelet-aggregation, granule-secretion, [Ca2+]i mobilization, integrin-αIIbβ3 activation were examined. Vasodilator-stimulated phosphoprotein (VASP) and MAPK expressions and levels of tyrosine phosphorylation of the glycoprotein VI (GPVI) signalling pathway components were also studied. G-Rp1 effects on arteriovenous shunt thrombus formation in rats or tail bleeding time and ex vivo coagulation time in mice were determined. KEY RESULT G-Rp1 markedly inhibited platelet aggregation induced by collagen, thrombin or ADP. While G-Rp1 elevated cAMP levels, it dose-dependently suppressed collagen-induced ATP-release, thromboxane secretion, p-selectin expression, [Ca2+]i mobilization and αIIbβ3 activation and attenuated p38MAPK and ERK2 activation. Furthermore, G-Rp1 inhibited tyrosine phosphorylation of multiple components (Fyn, Lyn, Syk, LAT, PI3K and PLCγ2) of the GPVI signalling pathway. G-Rp1 inhibited in vivo thrombus formation and ex vivo platelet aggregation and ATP secretion without affecting tail bleeding time and coagulation time, respectively. CONCLUSION AND IMPLICATIONS G-Rp1 inhibits collagen-induced platelet activation and thrombus formation through modulation of early GPVI signalling events, and this effect involves VASP stimulation, and ERK2 and p38-MAPK inhibition. These data suggest that G-Rp1 may have therapeutic potential for the treatment of cardiovascular diseases involving aberrant platelet activation. PMID

  14. Gallic acid ameliorates renal functions by inhibiting the activation of p38 MAPK in experimentally induced type 2 diabetic rats and cultured rat proximal tubular epithelial cells.

    PubMed

    Ahad, Amjid; Ahsan, Haseeb; Mujeeb, Mohd; Siddiqui, Waseem Ahmad

    2015-10-01

    Diabetic nephropathy (DN) is one of the leading causes of morbidity and mortality in diabetic patients that accounts for about 40% of deaths in type 2 diabetes. p38 mitogen activated protein kinase (p38 MAPK), a serine-threonine kinase, plays an important role in tissue inflammation and is known to be activated under conditions of oxidative stress and hyperglycemia. The role of p38 MAPK has been demonstrated in DN, and its inhibition has been suggested as an alternative approach in the treatment of DN. In the present study, we investigated the nephroprotective effects of an anti-inflammatory phenolic compound, gallic acid (GA, 3,4,5-trihydroxybenzoic acid), in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic wistar albino rats. GA (25 mg/kgbw and 50 mg/kgbw, p.o.) treatment for 16 weeks post induction of diabetes led to a significant reduction in the levels of blood glucose, HbA1c, serum creatinine, blood urea nitrogen and proteinuria as well as a significant reduction in the levels of creatinine clearance. GA significantly inhibited the renal p38 MAPK and nuclear factor kappa B (N-κB) activation as well as significantly reduced the levels of renal transforming growth factor beta (TGF-β) and fibronectin. Treatment with GA resulted in a significant reduction in the serum levels of proinflammatory cytokines viz. interleukin 1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α). Moreover, GA significantly lowered renal pathology and attenuated renal oxidative stress. In cultured rat NRK 52E proximal tubular epithelial cells, GA treatment inhibited high glucose induced activation of p38 MAPK and NF-κB as well as suppressed proinflammatory cytokine synthesis. The results of the present study provide in vivo and in vitro evidences that the p38 MAPK pathway plays an important role in the pathogenesis of DN, and GA attenuates the p38 MAPK-mediated renal dysfunction in HFD/STZ induced type 2 diabetic rats.

  15. Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

    PubMed Central

    Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

    2015-01-01

    Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

  16. Apoptosis Induction by the Total Flavonoids from Arachniodes exilis in HepG2 Cells through Reactive Oxygen Species-Mediated Mitochondrial Dysfunction Involving MAPK Activation

    PubMed Central

    Chen, Jing; Xiong, Chaomei; Wei, Han; Yin, Changchang; Ruan, Jinlan

    2014-01-01

    Arachniodes exilis is used as a folk medicine in China and proved to have antibacterial, anti-inflammatory, and sedative activities. In the present study, the antitumor effect of the total flavonoids of A. exilis (TFAE) against HepG2 cells was evaluated. The results showed that TFAE inhibited the growth of HepG2 cells in a dosage- and time-dependent manner. Flow cytometry and Hoechst 33342 fluorescence staining results showed that TFAE could significantly increase the apoptosis ratio of HepG2 cells, which is accompanied with increased intracellular reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). Western blotting indicated that TFAE downregulated the ratio of Bcl-2/Bax, increased cytochrome c release, and activated the caspases-3 and -9. Further analysis showed that TFAE stimulated the mitogen-activated protein kinase (MAPK). However, treatment with NAC (reactive oxygen species scavenger) and MAPK-specific inhibitors (SP600125 and SB203580) could reverse the changes of these apoptotic-related proteins. These results suggested that TFAE possessed potential anticancer activity in HepG2 cells through ROS-mediated mitochondrial dysfunction involving MAPK pathway. PMID:24976852

  17. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    SciTech Connect

    Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

    2010-07-20

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

  18. PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway

    PubMed Central

    Han, Hai-Bo; Gu, Jin; Ji, Deng-Bo; Li, Zhao-Wei; Zhang, Yuan; Zhao, Wei; Wang, Li-Min; Zhang, Zhi-Qian

    2014-01-01

    AIM: To investigate the role of pre-B-cell leukemia homeobox (PBX)3 in migration and invasion of colorectal cancer (CRC) cells. METHODS: We detected PBX3 expression in five cell lines and surgical specimens from 111 patients with CRC using real-time reverse transcription-polymerase chain reaction. We forced expression of PBX3 in low metastatic HT-29 and SW480 cells and knocked down expression of PBX3 in highly metastatic LOVO and HCT-8 cells. Wound healing and Boyden chamber assays were used to detect cell migration and invasion after altered expression of PBX3. Western blot was performed to detect the change of signaling molecule ERK1/2 following PBX3 overexpression. RESULTS: High level of PBX3 expression was correlated with the invasive potential of CRC cells, and significantly associated with lymph node invasion (P = 0.02), distant metastasis (P = 0.04), advanced TNM stage (P = 0.03) and poor overall survival of patients (P < 0.05). Ectopic expression of PBX3 in low metastatic cells was shown to promote migration and invasion, while inhibited PBX3 expression in highly metastatic cells suppressed migration and invasion. Furthermore, upregulation of phosphorylated extracellular signal-regulated kinase (ERK)1/2 was found to be one of the targeted molecules responsible for PBX3-induced CRC cell migration and invasion. CONCLUSION: PBX3 induces invasion and metastasis of CRC cells partially through activation of the MAPK/ERK signaling pathway. PMID:25561793

  19. Yes-associated protein 1 (YAP1) promotes human gallbladder tumor growth via activation of the AXL/MAPK pathway.

    PubMed

    Li, Maolan; Lu, Jianhua; Zhang, Fei; Li, Huaifeng; Zhang, Bingtai; Wu, Xiangsong; Tan, Zhujun; Zhang, Lin; Gao, Guofeng; Mu, Jiasheng; Shu, Yijun; Bao, Runfa; Ding, Qichen; Wu, Wenguang; Dong, Ping; Gu, Jun; Liu, Yingbin

    2014-12-28

    The transcriptional coactivator Yes-associated protein 1 (YAP1), a key regulator of cell proliferation and organ size in vertebrates, has been implicated in various malignancies. However, little is known about the expression and biological function of YAP1 in human gallbladder cancer (GBC). In this study we examined the clinical significance and biological functions of YAP1 in GBC and found that nuclear YAP1 and its target gene AXL were overexpressed in GBC tissues. We also observed a significant correlation between high YAP1 and AXL expression levels and worse prognosis. The depletion of YAP1 using lentivirus shRNAs significantly inhibited cell proliferation by inducing cell cycle arrest in S phase in concordance with the decrease of CDK2, CDC25A, and cyclin A, and resulted in increased cell apoptosis and invasive repression in GBC cell lines in vitro. Furthermore, knockdown of YAP1 also inhibited tumor growth in vivo. Additionally, we demonstrated that the activation of the AXL/MAPK pathway was involved in the oncogenic functions of YAP1 in GBC. These results demonstrated that YAP1 is a putative oncogene and represents a prognostic marker and potentially a novel therapeutic target for GBC.

  20. Celecoxib Inhibits the Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus through Down-Regulation of RTA Expression by Inhibiting the Activation of p38 MAPK.

    PubMed

    Chen, Jungang; Jiang, Liangyu; Lan, Ke; Chen, Xulin

    2015-05-01

    Kaposi's sarcoma associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV's lytic replication cycle is critical for the pathogenesis of KSHV-associated diseases. Despite recent progress in the development of treatments for KSHV associated malignancies, these therapies are not completely efficacious and cause side effects. Therefore, more effective therapies with antiviral agents against KSHV are urgently needed. In this study, we identified celecoxib as an antiviral agent against KSHV. Our data suggest that celecoxib inhibits the lytic activation of KSHV through the down-regulation of the expression of the lytic switch protein, replication and transcription activator (RTA), by inhibiting the activation of p38 MAPK. Therefore, celecoxib may provide a candidate inhibitor for the therapeutic research of KSHV-related malignancies.

  1. Baicalin Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9.

    PubMed

    Yan, Shuangquan; Wang, Yiran; Liu, Panpan; Chen, Ali; Chen, Mayun; Yao, Dan; Xu, Xiaomei; Wang, Liangxing; Huang, Xiaoying

    2016-01-01

    Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats, but the mechanism of this effect remains unclear. Thus, investigating the potential mechanism of this effect was the aim of the present study. Model rats that display hypoxic pulmonary hypertension and cor pulmonale under control conditions were successfully generated. We measured a series of indicators to observe the levels of pulmonary arterial hypertension, pulmonary arteriole remodeling, and right ventricular remodeling. We assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonary tissue homogenates using immunohistochemistry and western blot analyses, respectively. The matrix metalloproteinase- (MMP-) 9 protein and mRNA levels in the pulmonary arteriole walls were measured using immunohistochemistry and in situ hybridization. Our results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissue homogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls. This downregulation was accompanied by the attenuation of pulmonary hypertension, arteriole remodeling, and right ventricular remodeling. These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale, which are induced by chronic hypoxia, by downregulating the p38 MAPK/MMP-9 pathway. PMID:27688788

  2. Baicalin Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9

    PubMed Central

    Wang, Yiran; Chen, Ali; Chen, Mayun; Yao, Dan; Xu, Xiaomei; Wang, Liangxing

    2016-01-01

    Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats, but the mechanism of this effect remains unclear. Thus, investigating the potential mechanism of this effect was the aim of the present study. Model rats that display hypoxic pulmonary hypertension and cor pulmonale under control conditions were successfully generated. We measured a series of indicators to observe the levels of pulmonary arterial hypertension, pulmonary arteriole remodeling, and right ventricular remodeling. We assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonary tissue homogenates using immunohistochemistry and western blot analyses, respectively. The matrix metalloproteinase- (MMP-) 9 protein and mRNA levels in the pulmonary arteriole walls were measured using immunohistochemistry and in situ hybridization. Our results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissue homogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls. This downregulation was accompanied by the attenuation of pulmonary hypertension, arteriole remodeling, and right ventricular remodeling. These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale, which are induced by chronic hypoxia, by downregulating the p38 MAPK/MMP-9 pathway. PMID:27688788

  3. Baicalin Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension to Improve Hypoxic Cor Pulmonale by Reducing the Activity of the p38 MAPK Signaling Pathway and MMP-9

    PubMed Central

    Wang, Yiran; Chen, Ali; Chen, Mayun; Yao, Dan; Xu, Xiaomei; Wang, Liangxing

    2016-01-01

    Baicalin has a protective effect on hypoxia-induced pulmonary hypertension in rats, but the mechanism of this effect remains unclear. Thus, investigating the potential mechanism of this effect was the aim of the present study. Model rats that display hypoxic pulmonary hypertension and cor pulmonale under control conditions were successfully generated. We measured a series of indicators to observe the levels of pulmonary arterial hypertension, pulmonary arteriole remodeling, and right ventricular remodeling. We assessed the activation of p38 mitogen-activated protein kinase (MAPK) in the pulmonary arteriole walls and pulmonary tissue homogenates using immunohistochemistry and western blot analyses, respectively. The matrix metalloproteinase- (MMP-) 9 protein and mRNA levels in the pulmonary arteriole walls were measured using immunohistochemistry and in situ hybridization. Our results demonstrated that baicalin not only reduced p38 MAPK activation in both the pulmonary arteriole walls and tissue homogenates but also downregulated the protein and mRNA expression levels of MMP-9 in the pulmonary arteriole walls. This downregulation was accompanied by the attenuation of pulmonary hypertension, arteriole remodeling, and right ventricular remodeling. These results suggest that baicalin may attenuate pulmonary hypertension and cor pulmonale, which are induced by chronic hypoxia, by downregulating the p38 MAPK/MMP-9 pathway.

  4. The dynamics of MAPK inactivation at fertilization in mouse eggs

    PubMed Central

    Gonzalez-Garcia, Jose Raul; Bradley, Josephine; Nomikos, Michail; Paul, Laboni; Machaty, Zoltan; Lai, F. Anthony; Swann, Karl

    2014-01-01

    ABSTRACT Egg activation at fertilization in mammals is initiated by prolonged Ca2+ oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca2+ oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos–luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity. PMID:24741069

  5. Rosiglitazone attenuates NF-{kappa}B-dependent ICAM-1 and TNF-{alpha} production caused by homocysteine via inhibiting ERK{sub 1/2}/p38MAPK activation

    SciTech Connect

    Bai, Yong-Ping; Liu, Yu-Hui; Chen, Jia; Song, Tao; You, Yu; Tang, Zhen-Yan; Li, Yuan-Jian; Zhang, Guo-Gang . E-mail: xyzgg2006@sina.com

    2007-08-17

    Previous studies demonstrated an important interaction between nuclear factor-kappaB (NF-{kappa}B) activation and homocysteine (Hcy)-induced cytokines expression in endothelial cells and vascular smooth muscle cells. However, the underlying mechanism remains illusive. In this study, we investigated the effects of Hcy on NF-{kappa}B-mediated sICAM-1, TNF-{alpha} production and the possible involvement of ERK{sub 1/2}/p38MAPK pathway. The effects of rosiglitazone intervention were also examined. Our results show that Hcy increased the levels of sICAM-1 and TNF-{alpha} in cultured human umbilical vein endothelial cells (HUVECs) in a time- and concentration-dependent manner. This effect was significantly depressed by rosiglitazone and different inhibitors (PDTC, NF-{kappa}B inhibitor; PD98059, MEK inhibitor; SB203580, p38MAPK specific inhibitor; and staurosporine, PKC inhibitor). Next, we investigated the effect of Hcy on ERK{sub 1/2}/p38MAPK pathway and NF-{kappa}B activity in HUVECs. The results show that Hcy activated both ERK{sub 1/2}/p38MAPK pathway and NF-{kappa}B-DNA-binding activity. These effects were markedly inhibited by rosiglitazone as well as other inhibitors (SB203580, PD98059, and PDTC). Further, the pretreatment of staurosporine abrogated ERK{sub 1/2}/p38MAPK phosphorylation, suggesting that Hcy-induced ERK{sub 1/2}/p38MAPK activation is associated with PKC activity. Our results provide evidence that Hcy-induced NF-{kappa}B activation was mediated by activation of ERK{sub 1/2}/p38MAPK pathway involving PKC activity. Rosiglitazone reduces the NF-{kappa}B-mediated sICAM-1 and TNF-{alpha} production induced by Hcy via inhibition of ERK{sub 1/2}/p38MAPK pa0011thw.

  6. 4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

    SciTech Connect

    Lee, Seung J.; Kim, Chae E.; Yun, Mi R.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Shin, Hwa K.; Bae, Sun S.; Kim, Chi D.

    2010-01-15

    Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B{sub 4} (LTB{sub 4}) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT{sub 1} (cysLT{sub 1}) receptor antagonist, REV-5901 as well as a BLT{sub 1} receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB{sub 4} and cysLT (LTC{sub 4} and LTD{sub 4}) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB{sub 4} and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.

  7. Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells

    SciTech Connect

    Kim, Sung Hun; Yoo, Chong Il; Kim, Hui Taek; Park, Ji Yeon; Kwon, Chae Hwa; Keun Kim, Yong . E-mail: kim430@pusan.ac.kr

    2006-09-01

    The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.

  8. The activity of Mblk-1, a mushroom body-selective transcription factor from the honeybee, is modulated by the ras/MAPK pathway.

    PubMed

    Park, Jung-Min; Kunieda, Takekazu; Kubo, Takeo

    2003-05-16

    We previously identified a gene, termed Mblk-1, that encodes a putative transcription factor with two DNA-binding motifs expressed preferentially in the mushroom body of the honeybee brain, and its preferred binding sequence, termed Mblk-1-binding element (MBE) (Takeuchi, H., Kage, E., Sawata, M., Kamikouchi, A., Ohashi, K., Ohara, M., Fujiyuki, T., Kunieda, T., Sekimizu, K., Natori, S., and Kubo, T. (2001) Insect Mol Biol 10, 487-494; Park, J.-M., Kunieda. T., Takeuchi, H., and Kubo, T. (2002) Biochem. Biophys. Res. Commun. 291, 23-28). In the present study, the effect of Mblk-1 on transcription of genes containing MBE in Drosophila Schneider's Line 2 cells was examined using a luciferase assay. Mblk-1 expression transactivated promoters containing MBEs approximately 2-7-fold. Deletion experiments revealed that RHF2, the second DNA-binding domain of Mblk-1, was necessary for the transcriptional activity. Furthermore, mitogen-activated protein kinase (MAPK) phosphorylated Mblk-1 at Ser-444 in vitro, and the Mblk-1-induced transactivation was stimulated by phosphorylation of Ser-444 by the Ras/MAPK pathway in the luciferase assay. These results suggest that Mblk-1 is a transcription factor that might function in the mushroom body neuronal circuits downstream of the Ras/MAPK pathway in the honeybee brain.

  9. Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages.

    PubMed

    Banerjee, Somenath; Bose, Dipayan; Chatterjee, Nabanita; Das, Subhadip; Chakraborty, Sreeparna; Das, Tanya; Saha, Krishna Das

    2016-01-01

    Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future. PMID:26928472

  10. Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages

    PubMed Central

    Banerjee, Somenath; Bose, Dipayan; Chatterjee, Nabanita; Das, Subhadip; Chakraborty, Sreeparna; Das, Tanya; Saha, Krishna Das

    2016-01-01

    Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future. PMID:26928472

  11. p38MAPK activation is involved in androgen-independent proliferation of human prostate cancer cells by regulating IL-6 secretion

    SciTech Connect

    Shida, Yohei; Igawa, Tsukasa . E-mail: tigawa@net.nagasaki-u.ac.jp; Hakariya, Tomoaki; Sakai, Hideki; Kanetake, Hiroshi

    2007-02-16

    Increased levels of serum interleukin-6 (IL-6) are frequently observed in patients with advanced, hormone-refractory prostate cancer. However, the precise mechanism of IL-6 regulation is still largely unknown. Since prostate cancer gradually progresses to an androgen-independent state despite the stress caused by various therapeutic agents, we hypothesized the stress-activated protein kinases (SAPKs) involvement in androgen-independent growth or IL-6 secretion of prostate cancer cells. Using PC-3 and DU145 human prostate cancer cells, we analyzed the role of SAPKs in IL-6 mediated cell growth and found that the p38MAPK and JNK are involved in androgen-independent cancer cell growth. Furthermore, IL-6 secretion by PC-3 and DU145 cells was significantly suppressed by SAPKs inhibitor, especially by p38MAPK inhibitor SB203580, but not by JNK inhibitor SP600125 nor by MEK inhibitor, PD98059. These results raised the possibility that the IL-6 mediated androgen-independent proliferation of PC-3 and DU145 cells is regulated at least partly via SAPKs signaling pathway especially through p38MAPK activation.

  12. Isomenthone protects human dermal fibroblasts from TNF-α-induced death possibly by preventing activation of JNK and p38 MAPK.

    PubMed

    Jung, Eunsun; Byun, Sangyo; Kim, Seungbeom; Kim, Moohan; Park, Deokhoon; Lee, Jongsung

    2012-10-01

    Cell death evoked by tumor necrosis factor-α (TNF-α) is regulated by the TNF-α receptor-associated death domain containing protein, which interacts with and activates apoptotic proteases triggering cell death. c-Jun N-terminal kinase (JNK) and p38 MAPK, induce the apoptotic program and are indispensible early elements in stress-induced apoptosis that control the release of cytochrome c. Isomenthone is a constituent of the essential oil of Mentha arvensis L. and is used as a fragrance and flavor in the cosmetic, drug, and food industries. In this study, we investigated the protective effects of isomenthone against TNF-α-induced cell death and its mechanism in human dermal fibroblasts. To understand the cytoprotective role of isomenthone, MTT and terminal deoxynucleotidyl transferase dUTP nick end labeling assays for cell viability and enzyme-linked immunosorbent assay analysis for the mechanistic study were performed. We found that isomenthone inhibited the TNF-α-mediated reduction in cell viability and inhibited the increase in apoptosis under a serum-free condition. Isomenthone also blocked the JNK and p38 MAPK pathways and downstream apoptotic events. These results indicate that isomenthone has the potential to protect fibroblasts against TNF-α-induced cell death under a serum-deprived condition by blocking activation of the JNK and p38 MAPK pathways and downstream apoptotic events.

  13. Plantamajoside ameliorates lipopolysaccharide-induced acute lung injury via suppressing NF-κB and MAPK activation.

    PubMed

    Wu, Haichong; Zhao, Gan; Jiang, Kangfeng; Chen, Xiuying; Zhu, Zhe; Qiu, Changwei; Li, Chengye; Deng, Ganzhen

    2016-06-01

    Despite developments in the knowledge and therapy of acute lung injury in recent decades, mortality remains high, and there is usually a lack of effective therapy. Plantamajoside, a major ingredient isolated from Plantago asiatica L. (Plantaginaceae), has been reported to have potent anti-inflammatory properties. However, the effect of plantamajoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice has not been investigated. The present study aimed to reveal the potential mechanism responsible for the anti-inflammatory effects of plantamajoside on LPS-induced acute lung injury in mice and in RAW264.7 cells. The results of histopathological changes as well as the lung wet-to-dry ratio and myeloperoxidase (MPO) activity showed that plantamajoside ameliorated the lung injury that was induced by LPS. qPCR and ELISA assays demonstrated that plantamajoside suppressed the production of IL-1β, IL-6 and TNF-α in a dose-dependent manner. TLR4 is an important sensor in LPS infection. Molecular studies showed that the expression of TLR4 was inhibited by plantamajoside administration. Further study was conducted on nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) using pathways using western blots. The results showed that plantamajoside inhibited the phosphorylation of IκBα, p65, p38, JNK and ERK. All results indicated that plantamajoside has protective effect on LPS-induced ALI in mice and in RAW264.7 cells. Thus, plantamajoside may be a potential therapy for the treatment of pulmonary inflammation. PMID:27089391

  14. Plantamajoside ameliorates lipopolysaccharide-induced acute lung injury via suppressing NF-κB and MAPK activation.

    PubMed

    Wu, Haichong; Zhao, Gan; Jiang, Kangfeng; Chen, Xiuying; Zhu, Zhe; Qiu, Changwei; Li, Chengye; Deng, Ganzhen

    2016-06-01

    Despite developments in the knowledge and therapy of acute lung injury in recent decades, mortality remains high, and there is usually a lack of effective therapy. Plantamajoside, a major ingredient isolated from Plantago asiatica L. (Plantaginaceae), has been reported to have potent anti-inflammatory properties. However, the effect of plantamajoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice has not been investigated. The present study aimed to reveal the potential mechanism responsible for the anti-inflammatory effects of plantamajoside on LPS-induced acute lung injury in mice and in RAW264.7 cells. The results of histopathological changes as well as the lung wet-to-dry ratio and myeloperoxidase (MPO) activity showed that plantamajoside ameliorated the lung injury that was induced by LPS. qPCR and ELISA assays demonstrated that plantamajoside suppressed the production of IL-1β, IL-6 and TNF-α in a dose-dependent manner. TLR4 is an important sensor in LPS infection. Molecular studies showed that the expression of TLR4 was inhibited by plantamajoside administration. Further study was conducted on nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) using pathways using western blots. The results showed that plantamajoside inhibited the phosphorylation of IκBα, p65, p38, JNK and ERK. All results indicated that plantamajoside has protective effect on LPS-induced ALI in mice and in RAW264.7 cells. Thus, plantamajoside may be a potential therapy for the treatment of pulmonary inflammation.

  15. Chlamydial plasmid-encoded protein pORF5 induces production of IL-1β and IL-18 via NALP3 inflammasome activation and p38 MAPK pathway

    PubMed Central

    Cao, Wenjuan; Zou, Yan; Su, Shengmei; He, Zhansheng; Liu, Yan; Huang, Qiulin; Li, Zhongyu

    2015-01-01

    The pathogenesis of Chlamydia-induced inflammation is poorly understood. pORF5 is the only secreted protein encoded by Chlamydial plasmid. This study aims to investigate the effects of pORF5 on the production of interleukin-1β (IL-1β) and interleukin-18 (IL-18) and the underlying mechanisms of these effects. THP-1 (a human acute monocytic leukemia cell line) cells were stimulated by pORF5 with or without pretreatment with Natch domain, Leucine-rich repeat and PYD-containing protein 3 (NALP3) siRNA, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) siRNA, cysteine aspartate-specific protease-1 (caspase-1) specific inhibitor and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. IL-1β, IL-18 and caspase-1 expression was detected through both ELISA and qRT-PCR. NALP3 and ASC expression was detected by qRT-PCR. The expression of caspase-1 and phosphorylated-p38 MAPK was detected by western blot analysis. pORF5 induced IL-1β, IL-18, caspase-1 and NALP3 inflammasome expression in THP-1 cells. Caspase-1 inhibitor significantly reduced pORF5-induced IL-1β and IL-18 expression. The siRNAs for NALP3 inflammasome significantly reduced pORF5-induced IL-1β, IL-18 and caspase-1 expression. Furthermore, p38 MAPK inhibitor significantly reduced pORF5-induced IL-1β, IL-18, caspase-1 and NALP3 inflammasome expression. pORF5 could induce production of IL-1β and IL-18 via NALP3 inflammasome activation and p38MAPK pathway. pORF5 protein might play an important role in Chlamydia pathogenesis. This study provides a new insight into the molecular pathogenesis of Chlamydial diseases. PMID:26884953

  16. Nanosized titanium dioxide resulted in the activation of TGF-β/Smads/p38MAPK pathway in renal inflammation and fibration of mice.

    PubMed

    Hong, F; Wu, N; Ge, Y; Zhou, Y; Shen, T; Qiang, Q; Zhang, Q; Chen, M; Wang, Y; Wang, L; Hong, J

    2016-06-01

    Titanium dioxide nanoparticles (TiO2 NPs) have been demonstrated to damage the kidneys. However, whether chronic nephritis leads to renal fibration or the fibrosis is associated with the activation of TGF-β/Smads/p38MAPK pathway caused by TiO2 NPs exposure is not well understood. Forty male mice were separately exposed to 0, 2.5, 5, or 10 mg/kg body weight TiO2 NPs for 6 months. Renal biochemical functions and levels of TGF-β/Smads/p38MAPK pathway-related markers and extracellular matrix (ECM) expression in the kidneys were investigated. The findings showed that subchronic TiO2 NPs exposure increased levels of urinary creatisix (Cr), N-acetyl-glucosaminidase, and vanin-1, resulted in severe renal inflammation and fibration. Furthermore, TiO2 NP exposure upregulated expression of transforming growth factor-β1 (TGF-β1, 0.07- to 2.72-fold), Smad2 (0.42- to 1.63-fold), Smad3 (0.02- to 1.94-fold), ECM (0.15- to 2.75-fold), α-smooth muscle actin (0.14- to 3.06-fold), p38 mitogen-activated protein kinase (p38MAPK, 0.11- to 3.78-fold), and nuclear factor-κB (0.4- to 2.27-fold), and downregulated Smad7 (0.05- to 0.61-fold) expression in mouse kidney. Subchronic TiO2 NPs exposure induced changes of renal characteristics towards inflammation and fibration may be mediated via TGF-β/Smads/p38MAPK pathway, and the uses of TiO2 NPs should be carried out cautiously, especially in humans. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1452-1461, 2016.

  17. Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway.

    PubMed Central

    Geppert, T. D.; Whitehurst, C. E.; Thompson, P.; Beutler, B.

    1994-01-01

    BACKGROUND: Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. MATERIALS AND METHODS: Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. RESULTS: In RAW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as well as derepression of the translational blockade normally imposed by the TNF 3'-untranslated region. A constitutively active form of raf-1 (raf-BXB) was found to augment, but not replace, the LPS signal. In LPS-insensitive cells (RAW 264.7 x NIH 3T3 fusion hybrid cells and primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. CONCLUSIONS: The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages

  18. Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation.

    PubMed

    Sabbagh, W; Flatauer, L J; Bardwell, A J; Bardwell, L

    2001-09-01

    Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover. PMID:11583629

  19. Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation

    PubMed Central

    NaveenKumar, Somanathapura K.; Thushara, Ram M.; Sundaram, Mahalingam S.; Hemshekhar, Mahadevappa; Paul, Manoj; Thirunavukkarasu, Chinnasamy; Basappa; Nagaraju, Ganesh; Raghavan, Sathees C.; Girish, Kesturu S.; Kemparaju, Kempaiah; Rangappa, Kanchugarakoppal S.

    2015-01-01

    Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation. PMID:26459859

  20. Mobile phone electromagnetic radiation activates MAPK signaling and regulates viability in Drosophila.

    PubMed

    Lee, Kyu-Sun; Choi, Jong-Soon; Hong, Sae-Yong; Son, Tae-Ho; Yu, Kweon

    2008-07-01

    Mobile phones are widely used in the modern world. However, biological effects of electromagnetic radiation produced by mobile phones are largely unknown. In this report, we show biological effects of the mobile phone 835 MHz electromagnetic field (EMF) in the Drosophila model system. When flies were exposed to the specific absorption rate (SAR) 1.6 W/kg, which is the proposed exposure limit by the American National Standards Institute (ANSI), more than 90% of the flies were viable even after the 30 h exposure. However, in the SAR 4.0 W/kg strong EMF exposure, viability dropped from the 12 h exposure. These EMF exposures triggered stress response and increased the production of reactive oxygen species. The EMF exposures also activated extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling, but not p38 kinase signaling. Interestingly, SAR 1.6 W/kg activated mainly ERK signaling and expression of an anti-apoptotic gene, whereas SAR 4.0 W/kg strongly activated JNK signaling and expression of apoptotic genes. In addition, SAR 4.0 W/kg amplified the number of apoptotic cells in the fly brain. These findings demonstrate that the exposure limit on electromagnetic radiation proposed by ANSI triggered ERK-survival signaling but the strong electromagnetic radiation activated JNK-apoptotic signaling in Drosophila.

  1. Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

    PubMed

    Yang, Long-Long; Zhou, Yan; Tian, Wei-Dong; Li, Hai-Juan; Kang-Chu-Li; Miao, Xia; An, Guang-Zhou; Wang, Xiao-Wu; Guo, Guo-Zhen; Ding, Gui-Rong

    2016-01-01

    Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1β, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1β, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process. PMID:26688329

  2. Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

    PubMed

    Yang, Long-Long; Zhou, Yan; Tian, Wei-Dong; Li, Hai-Juan; Kang-Chu-Li; Miao, Xia; An, Guang-Zhou; Wang, Xiao-Wu; Guo, Guo-Zhen; Ding, Gui-Rong

    2016-01-01

    Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1β, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1β, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process.

  3. Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

    PubMed Central

    2012-01-01

    Background In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4) activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria) can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1), which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs). Results We demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88), Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2), and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], c-Src (PP1), p38 MAPK (SB202190), and p300 (GR343). LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody. Conclusions In HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may reduce monocyte

  4. Neomorphic Mutations in PIK3R1 Confer Sensitivity to MAPK Inhibitors due to Activation of ERK and JNK Pathways | Office of Cancer Genomics

    Cancer.gov

    In a recent publication in Cancer Cell, CTD2 investigators discovered that a known cancer-associated gain-of-function alteration in phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) results in novel protein activity that confers sensitivity to mitogen-activated protein kinase (MAPK) inhibitors. The PIK3R1 gene encodes the p85α regulatory subunit of PIK3. Under normal conditions, p85α suppresses PIK3 mediated activation of downstream pathways that promote cell growth and survival.

  5. Phloroglucinol Reduces Photodamage in Hairless Mice via Matrix Metalloproteinase Activity Through MAPK Pathway.

    PubMed

    Im, A-Rang; Nam, Kung-Woo; Hyun, Jin Won; Chae, Sungwook

    2016-01-01

    We investigated the photoprotective activity of phloroglucinol on ultraviolet B (UVB)-induced deleterious effects in hairless mice in vivo. To assess the photoprotective effect of phloroglucinol, phloroglucinol-treated HR-1 hairless male mice were exposed to UVB irradiation. The inhibitory activity of phloroglucinol on wrinkle formation was determined by analysis of skin replicas, epidermal thickness based on histological examination and collagen damage. Matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase (TIMP) mRNA levels were measured by real-time PCR. UVB induced transcription of proinflammatory cytokines, including interleukin-1 beta (IL-1β, IL-6) and IL-8 (IL-8). The protective effects of phloroglucinol on UVB-induced skin photoaging were examined by measuring protein levels of MMPs and mitogen-activated protein (MAP) kinases. The results of these experiments suggest that phloroglucinol has a significant beneficial effect on the barrier function of the skin. In hairless mice, signs of photoaging and photodamage, including coarse wrinkle formation, epidermal thickness and elastic fiber degeneration, were reduced in severity by phloroglucinol application. The phloroglucinol-treated group showed remarkably decreased mRNA levels of MMP-1, MMP-9 and inflammatory cytokines in comparison with those of the UVB-induced group. Oral administration of phloroglucinol attenuated phosphorylation of MAP kinases, including extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38. PMID:26537624

  6. The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

    PubMed

    Miguel-Rojas, Cristina; Hera, Concepcion

    2016-01-01

    F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.

  7. Status report of RMS active damping augmentation

    NASA Technical Reports Server (NTRS)

    Gilbert, Mike; Demeo, Martha E.

    1993-01-01

    A status report of Remote Manipulator System (RMS) active damping augmentation is presented. Topics covered include: active damping augmentation; benefits of RMS ADA; simulated payload definition; sensor and actuator definition; ADA control law design; Shuttle Engineering Simulator (SES) real-time simulation; and astronaut evaluation.

  8. Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis.

    PubMed

    Sun, Yan; Zhang, Lingling; Zhang, Meiwei; Li, Ruojiao; Li, Yangping; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

    2016-07-01

    Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr kinases that play a vital role in innate immune responses by converting extracellular stimuli into a wide range of cellular responses. Although MAPKs have been extensively studied in various vertebrates and invertebrates, our current understanding of MAPK signaling cascade in scallop is in its infancy. In this study, three MAPK genes (PyERK, PyJNK, and Pyp38) were identified from Yesso scallop Patinopecten yessoensis. The open reading frame of PyERK, PyJNK, and Pyp38 was 1104, 1227, and 1104 bp, encoding 367, 408, and 367 amino acids, respectively. Conservation in some splicing sites was revealed across the three PyMAPKs, suggesting the common descent of MAPKs genes. The expression profiles of PyMAPKs over the course of ten different developmental stages showed that they had different expression patterns. In adult scallops, PyMAPKs were primarily expressed in muscles, hemocytes, gill, and mantle. To gain insights into their role in innate immunity, we investigated their expression profiles after infection with Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Vibrio anguillarum). Significant difference in gene expression was only found in PyERK and PyJNK, but not Pyp38, suggesting Pyp38 may not participate in immune response to bacterial infection. Besides, PyERK and PyJNK exhibited more drastic change against the invasion of V. anguillarum than M. luteus, suggesting they could be more sensitive to Gram-negative bacteria than Gram-positive bacteria. This study provides valuable resource for elucidating the role of MAPK signal pathway in bivalve innate immune response. PMID:27155450

  9. Angiotensin II-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells.

    PubMed

    Morales, María Gabriela; Vazquez, Yaneisi; Acuña, María José; Rivera, Juan Carlos; Simon, Felipe; Salas, José Diego; Alvarez Ruf, Joel; Brandan, Enrique; Cabello-Verrugio, Claudio

    2012-11-01

    Fibrotic disorders are typically characterised by excessive connective tissue and extracellular matrix (ECM) deposition that preclude the normal healing of different tissues. Several skeletal muscle dystrophies are characterised by extensive fibrosis. Among the factors involved in skeletal muscle fibrosis is angiotensin II (Ang-II), a key protein of the renin-angiotensin system (RAS). We previously demonstrated that myoblasts responded to Ang-II by increasing the ECM protein levels mediated by AT-1 receptors, implicating an Ang-II-induced reactive oxygen species (ROS) by a NAD(P)H oxidase-dependent mechanism. In this paper, we show that in myoblasts, Ang-II induced the increase of transforming growth factor beta 1 (TGF-β1) and connective tissue growth factor (CTGF) expression through its AT-1 receptor. This effect is dependent of the NAD(P)H oxidase (NOX)-induced ROS, as indicated by a decrease of the expression of both pro-fibrotic factors when the ROS production was inhibited via the NOX inhibitor apocynin. The increase in pro-fibrotic factors levels was paralleled by enhanced p38MAPK and ERK1/2 phosphorylation in response to Ang-II. However, only the p38MAPK activity was critical for the Ang-II-induced fibrotic effects, as indicated by the decrease in the Ang-II-induced TGF-β1 and CTGF expression and fibronectin levels by SB-203580, an inhibitor of the p38MAPK, but not by U0126, an inhibitor of ERK1/2 phosphorylation. Furthermore, we showed that the Ang-II-dependent p38MAPK activation, but not the ERK1/2 phosphorylation, was necessary for the NOX-derived ROS. In addition, we demonstrated that TGF-β1 expression was required for the Ang-II-induced pro-fibrotic effects evaluated by using SB-431542, an inhibitor of TGF-βRI kinase activity, and by knocking down TGF-β1 levels by shRNA technique. These results strongly suggest that the fibrotic response to Ang-II is mediated by the AT-1 receptor and requires the p38MAPK phosphorylation, NOX-induced ROS, and TGF

  10. Weight Status and Physical Activity

    PubMed Central

    Wang, Helen; Blanco, Estela; Algarín, Cecilia; Peirano, Patricio; Burrows, Raquel; Reyes, Marcela; Wing, David; Godino, Job G.; Gahagan, Sheila

    2016-01-01

    We tested the independent and combined influence of overweight/obesity and meeting moderate to vigorous physical activity (MVPA) guidelines (≥60 minutes per day) on cardiometabolic risk factors among healthy adolescents. We measured anthropometry, blood pressure, fasting lipids, and activity by accelerometer in 223 adolescents. They were categorized as overweight/obese versus normal weight and meeting the World Health Organization guidelines for MVPA per day. Adolescents were 16.8 years, 41% overweight/obese, 30% met MVPA guidelines, 50% low high-density lipoprotein, 22% high triglycerides, 12% high blood pressure, and 6% high fasting glucose. Controlling for sex, overweight/obese adolescents who did not meet MVPA guidelines had 4.0 and 11.9 increased odds for elevated triglycerides and systolic blood pressure, respectively, compared to normal weight adolescents who met MVPA guidelines. Overweight/obese and normal weight adolescents who met MVPA guidelines did not differ in cardiometabolic risk factors. Among overweight/obese adolescents, being physically active attenuated the likelihood of high triglycerides and systolic blood pressure. PMID:27803943

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

    PubMed Central

    Rousseau, Simon; Martel, Guy

    2016-01-01

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

  12. Contribution of S6K1/MAPK Signaling Pathways in the Response to Oxidative Stress: Activation of RSK and MSK by Hydrogen Peroxide

    PubMed Central

    Schneider, Taiane; Bonan, Carla Denise; Bartrons, Ramon; Ventura, Francesc; Rodrigues de Oliveira, Jarbas; Rosa, Jose Luis

    2013-01-01

    Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals’ knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2. PMID:24058693

  13. Critical role of the transient activation of p38 MAPK in the etiology of skeletal muscle insulin resistance induced by low-level in vitro oxidant stress

    PubMed Central

    Diamond-Stanic, Maggie K.; Marchionne, Elizabeth M.; Teachey, Mary K.; Durazo, David E.; Kim, John S.; Henriksen, Erik J.

    2011-01-01

    Increased cellular exposure to oxidants may contribute to the development of insulin resistance and type 2 diabetes. Skeletal muscle is the primary site of insulin-dependent glucose disposal in the body; however, the effects of oxidative stress on insulin signaling and glucose transport activity in mammalian skeletal muscle are not well understood. We therefore studied the effects of a low-level in vitro oxidant stress (30–40 μM H2O2) on basal and insulin-stimulated (5 mU/ml) glucose transport activity and insulin signaling at 2, 4, and 6 hr in isolated rat soleus muscle. H2O2 increased basal glucose transport activity at 2 and 4 hr, but not at 6 hr. This lowlevel oxidant stress significantly impaired insulin-stimulated glucose transport activity at all time points, and was associated with inhibition of insulin-stimulated phosphorylation of Akt Ser473 and GSK-3β Ser9. In the presence of insulin, H2O2 decreased total protein expression of IRS-1 at 6 hr and IRS-2 at 4 and 6 hr. Phosphorylation of p38 MAPK Thr180/Tyr182 was transiently increased by H2O2 in the presence and absence of insulin at 2 and 4 hr, but not at 6 hr. Selective inhibition of p38 MAPK with A304000 partially rescued the H2O2-induced reduction in insulin-stimulated glucose transport activity. These results indicate that direct in vitro exposure of isolated mammalian skeletal muscle to a low-level oxidant stress impairs distal insulin signaling and insulin-stimulated glucose transport activity, at least in part, due to a p38 MAPK-dependent mechanism. PMID:21241662

  14. Centipedegrass extract induces apoptosis through the activation of caspases and the downregulation of PI3K/Akt and MAPK phosphorylation in leukemia cells.

    PubMed

    Bai, Hyoung-Woo; Badaboina, Srilatha; Park, Chul-Hong; Choi, Bo Yun; Na, Yun Hee; Chung, Byung Yeoup

    2015-02-01

    Acute lymphoblastic leukemia (ALL), which involves the blood and bone marrow, is the most common type of cancer in children younger than 5 years of age. Previous studies have investigated the effects of centipedegrass extract (CGE), which is mainly composed of maysin and its derivatives, and have demonstrated that it has various biological activities, including antioxidant and anti‑inflammatory activities, pancreatic lipase inhibitory activity, anti-adipogenic activity and insecticidal activity. To the best of our knowledge, this study is the first to investigate the anticancer effects of CGE in ALL cell lines and to elucidate the mechanisms underlying these effects. Cell viability was measured by thiazolyl blue tetrazolium blue (MTT) assay. Apoptosis, cell cycle progression and mitochondrial membrane potential (∆Ψm) were determined by flow cytometry. The effects of CGE on the phosphatidylinositol 3‑kinase (PI3K)/Akt pathway and mitogen‑activated protein kinases (MAPKs) were assessed by immunoblotting. PI3K, MAPK and caspase inhibitors were used to further confirm the molecular mechanisms involved. Our results clearly demonstrated that the proliferation of the ALL cells was significantly inhibited by CGE in a dose‑dependent manner. Apoptosis was accompanied by the induction of significant G1 cell cycle arrest. The resulting alteration of the ∆Ψm increased the activity of caspase‑3/7. The induction of apoptosis was enhanced by the combined treatment of CGE with a PI3K inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, whereas the CGE‑induced apoptosis was inhibited in the presence of caspase inhibitors, such as z‑VAD‑fmk and z‑IETD‑fmk. Furthermore, CGE inhibited PI3K activity by decreasing the levels of phosphorylated (p‑)Akt, p‑BAD, and Bcl‑2 together with the levels of MAPKs, including p‑ERK and p‑JNK, but demonstrated no effects on p38 MAPK. Thus, our data suggest that CGE may be a novel natural

  15. The azetidine derivative, KHG26792 protects against ATP-induced activation of NFAT and MAPK pathways through P2X7 receptor in microglia.

    PubMed

    Kim, Eun-A; Cho, Chang Hun; Kim, Jiae; Hahn, Hoh-Gyu; Choi, Soo Young; Yang, Seung-Ju; Cho, Sung-Woo

    2015-12-01

    Azetidine derivatives are of interest for drug development because they may be useful therapeutic agents. However, their mechanisms of action remain to be completely elucidated. Here, we have investigated the effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on ATP-induced activation of NFAT and MAPK through P2X7 receptor in the BV-2 mouse microglial cell line. KHG26792 decreased ATP-induced TNF-α release from BV-2 microglia by suppressing, at least partly, P2X7 receptor stimulation. KHG26792 also inhibited the ATP-induced increase in IL-6, PGE2, NO, ROS, CXCL2, and CCL3. ATP induced NFAT activation through P2X7 receptor, with KHG26792 reducing the ATP-induced NFAT activation. KHG26792 inhibited an ATP-induced increase in iNOS protein and ERK phosphorylation. KHG26792 prevented an ATP-induced increase in MMP-9 activity through the P2X7 receptor as a result of degradation of TIMP-1 by cathepsin B. Our data provide mechanistic insights into the role of KHG26792 in the inhibition of TNF-α produced via P2X7 receptor-mediated activation of NFAT and MAPK pathways in ATP-treated BV-2 cells. This study highlights the potential use of KHG26792 as a therapeutic agent for the many diseases of the CNS related to activated microglia.

  16. Paeonol suppresses oxidized low-density lipoprotein induced endothelial cell apoptosis via activation of LOX-1/p38MAPK/NF-κB pathway.

    PubMed

    Bao, Mei-Hua; Zhang, Yi-Wen; Zhou, Hong-Hao

    2013-03-27

    Paeonol is an active compound isolated from traditional Chinese medicine, and has been shown to have anti-atherosclerosis, anti-inflammatory, antioxidant effects. The present investigation was undertaken to determine the suppression effects of paeonol on oxidized low-density lipoprotein (ox-LDL) induced endothelial cell line HUVEC apoptosis and to uncover some of the underlying mechanisms of these effects. Cell viability and lactate dehydrogenase (LDH) were measured to evaluate the cell injuries. Apoptosis was evaluated by Hoechst 33342 staining and flow cytometry. Intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Real-time PCR was used to confirm the expression of LOX-1 mRNA. Western blotting was used to evaluate the protein expression of LOX-1 and Bcl-2, as well as caspase-3 cleavage, p38-mitogen-activated protein kinase (p38MAPK) phosphorylation. NF-κB nuclear translocation was detected by Western blotting and immunofluorescence. Caspase-3 activity was measured using a colorimetric protease assay kit. The results showed that ox-LDL significantly decreased cell viability and increased the LDH release, as well as the apoptotic rate (P<0.01). Pre-treatment of paeonol resulted in remarkable increase of cell viability, decrease of LDH release and cell apoptosis in a concentration-dependent manner. Besides, ox-LDL caused the up-regulation of LOX-1, the down-regulation of Bcl-2, the phosphorylation of p38MAPK, the translocation of NF-κB and the activation of caspase-3. Paeonol pre-treatment reversed these effects introduced by ox-LDL. Moreover, paeonol also showed its inhibition effects on ox-LDL induced ROS overproduction. These results indicate the preventive effects of paeonol on ox-LDL induced endothelial cell apoptosis. The effects might, at least partly, be obtained via inhibition of LOX-1-ROS- p38MAPK-NF-κB signaling pathway.

  17. β-Elemonic acid inhibits the cell proliferation of human lung adenocarcinoma A549 cells: The role of MAPK, ROS activation and glutathione depletion.

    PubMed

    Wu, Tsu-Tuan; Lu, Chien-Lin; Lin, Hen-I; Chen, Bing-Fang; Jow, Guey-Mei

    2016-01-01

    β-elemonic acid, a known triterpene, exhibits anti-inflammatory effects, yet research on the pharmacological effects of β-elemonic acid is rare. We investigated the anticancer effects and the related molecular mechanisms of β-elemonic acid on human non-small cell lung cancer (NSCLC) A549 cells. The effects of β-elemonic acid on the growth of A549 cells were studied using a 3-(4,5)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected using Annexin V staining. The effect of β-elemonic acid on the cell cycle of A549 cells was assessed using the propidium iodide method. The change in reactive oxygen species (ROS) was detected using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay with microscopic examination. The expression levels of Bcl-2 family proteins, mitogen-activated protein kinase (MAPK) family proteins and cyclooxygenase 2 (COX-2) were detected using western blot analysis. Our data revealed that β-elemonic acid strongly induced human A549 lung cancer cell death in a dose- and time-dependent manner as determined by the MTT assay. β-elemonic acid-induced cell death was considered to be apoptotic when the phosphatidylserine exposure was observed using Annexin V staining. The death of human A549 lung cancer cells was caused by apoptosis induced by activation of ROS activity, increase in the sub-G1 proportion, downregulation of Bcl-2 expression, upregulation of Bax expression and inhibition of the MAPK signaling pathways. These results clearly demonstrated that β-elemonic acid inhibits proliferation by inducing hypoploid cells and cell apoptosis. Moreover, the anticancer effects of β-elemonic acid were related to the MAPK signaling pathway, ROS activation and glutathione depletion in human A549 lung cancer cells.

  18. Combined Effects of Cadmium and UVB Radiation on Sea Urchin Embryos: Skeleton Impairment Parallels p38 MAPK Activation and Stress Genes Overexpression.

    PubMed

    Bonaventura, Rosa; Russo, Roberta; Zito, Francesca; Matranga, Valeria

    2015-05-18

    Human and natural activities release many pollutants in the marine environment. The mixture of pollutants can affect many organisms concurrently. We used Paracentrotus lividus as a model to analyze the effects on signal transduction pathways and stress gene expression in embryos exposed continuously to double stress, i.e., cadmium (Cd) from fertilization and UVB at cleavage (Cd/UVB-embryos). By microscopical inspection, we evaluated embryonic morphology after 72 h of development. Tissue-specific markers were used to assess mesoderm differentiation by immunofluorescence. We analyzed p38MAPK, ERK1/2, and JNK activation by Western blot and mRNA profiles of Pl-MT, Pl-14-3-3epsilon, and Pl-jun genes by real-time quantitative polymerase chain reaction (qPCR) and the localization of their transcripts by whole mount in situ hybridization (WMISH). We found that the Cd/UVB combined exposure induced morphological malformations in 76% of pluteus embryos, mainly affecting the development of the skeleton, including the normal branching of skeletal roads. In Cd/UVB-embryos, p38MAPK was activated 1 h after UVB exposure and a remarkable overexpression of the Pl-MT, Pl-14.3.3epsilon, and Pl-jun genes 24 h after UVB exposure. Pl-MT and Pl-14.3.3epsilon mRNAs were misexpressed as they were localized in a position different from that observed in wild-type embryos, i.e., the intestine. On the contrary, Pl-jun mRNA has remained localized in the skeletogenic cells despite their displacement in exposed embryos. In conclusion, Cd/UVB exposure affected skeletal patterning producing alternative morphologies in which p38MAPK activation and Pl-MT, Pl-14.3.3epsilon, and Pl-jun gene overexpression seem linked to a protective role against the stress response induced by Cd/UVB.

  19. Total flavonoids of Hedyotis diffusa Willd inhibit inflammatory responses in LPS-activated macrophages via suppression of the NF-κB and MAPK signaling pathways

    PubMed Central

    CHEN, YUNLONG; LIN, YANYAN; LI, YACHAN; LI, CANDONG

    2016-01-01

    Nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways play a central role in inflammatory responses. Total flavonoids of Hedyotis diffusa Willd (TFHDW) are active compounds derived from Hedyotis diffusa Willd, which has been long used in Chinese traditional medicine for the treatment of various inflammatory diseases, including ulcerative colitis and bronchitis; however, the precise mechanisms underlying the effects of TFHDW are largely unknown. In the present study, the anti-inflammatory effect of TFHDW was evaluated and the underlying molecular mechanisms were investigated in an in vitro inflammatory model comprising lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The results indicated that TFHDW inhibited the inflammatory response as it significantly reduced the LPS-induced expression of pro-inflammatory nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in a concentration-dependent manner, without causing cytotoxicity. In addition, the mRNA expression of inducible nitric oxide synthase, TNF-α, IL-6 and IL-1β was suppressed by treatment with TFHDW in LPS-stimulated RAW 264.7 cells. Moreover, TFHDW treatment significantly inhibited the LPS-induced activation of NF-κB via the suppression of inhibitor of κB (IκB) phosphorylation, and reduced the phosphorylation of MAPK signaling molecules (p38, c-Jun N-terminal protein kinase and extracellular signal-regulated kinase 1/2), which resulted in the inhibition of cytokine expression. These findings suggest that TFHDW exerted anti-inflammatory activity via suppression of the NF-κB and MAPK signaling pathways. PMID:26998046

  20. Thymoquinone prevents RANKL-induced osteoclastogenesis activation and osteolysis in an in vivo model of inflammation by suppressing NF-KB and MAPK Signalling.

    PubMed

    Thummuri, Dinesh; Jeengar, Manish Kumar; Shrivastava, Shweta; Nemani, Harishankar; Ramavat, Ravindar Naik; Chaudhari, Pradip; Naidu, V G M

    2015-09-01

    Osteoclasts are multinuclear giant cells responsible for bone resorption in inflammatory bone diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Because of deleterious side effects with currently available drugs the search continues for novel effective and safe therapies. Thymoquinone (TQ), the major bioactive component of Nigella sativa has been investigated for its anti-inflammatory, antioxidant and anticancer activities. However, its effects in osteoclastogenesis have not been reported. In the present study we show for the first time that TQ inhibits nuclear factor-KB ligand (RANKL) induced osteoclastogenesis in RAW 264.7 and primary bone marrow derived macrophages (BMMs) cells. RANKL induced osteoclastogenesis is associated with increased expression of multiple transcription factors via activation of NF-KB, MAPKs signalling and reactive oxygen species (ROS). Mechanistically TQ blocked the RANKL induced NF-KB activation by attenuating the phosphorylation of IkB kinase (IKKα/β). Interestingly, in RAW 264.7 cells TQ inhibited the RANKL induced phosphorylation of MAPKs and mRNA expression of osteoclastic specific genes such as TRAP, DC-STAMP, NFATc1 and c-Fos. In addition, TQ also decreased the RANKL stimulated ROS generation in macropahges (RAW 264.7) and H2O2 induced ROS generation in osteoblasts (MC-3T3-E1). Consistent with in vitro results, TQ inhibited lipopolysaccharide (LPS) induced bone resorption by suppressing the osteoclastogenesis. Indeed, micro-CT analysis showed that bone mineral density (BMD) and bone architecture parameters were positively modulated by TQ. Taken together our data demonstrate that TQ has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-KB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

  1. Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice

    SciTech Connect

    Kim, Young Nam; Kim, Dae Won; Jo, Hyo Sang; Shin, Min Jea; Ahn, Eun Hee; Ryu, Eun Ji; Yong, Ji In; Cha, Hyun Ju; Kim, Sang Jin; Yeo, Hyeon Ji; Youn, Jong Kyu; Hwang, Jae Hyeok; Jeong, Ji-Heon; Kim, Duk-Soo; Cho, Sung-Woo; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2015-07-15

    Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases. - Highlights: • Transduced Tat-CBR1 reduces LPS-induced inflammatory mediators and cytokines. • Tat-CBR1 inhibits MAPK and NF-κB activation. • Tat-CBR1 ameliorates inflammation response in vitro and in vivo. • Tat-CBR1 may be useful as potential therapeutic agent for inflammation.

  2. Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

    PubMed

    Gendron, L; Laflamme, L; Rivard, N; Asselin, C; Payet, M D; Gallo-Payet, N

    1999-09-01

    In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.

  3. Anti-Food Allergic Activity of Sulfated Polysaccharide from Gracilaria lemaneiformis is Dependent on Immunosuppression and Inhibition of p38 MAPK.

    PubMed

    Liu, Qing-Mei; Yang, Yang; Maleki, Soheila J; Alcocer, Marcos; Xu, Sha-Sha; Shi, Chao-Lan; Cao, Min-Jie; Liu, Guang-Ming

    2016-06-01

    Polysaccharides from Gracilaria lemaneiformis in particular possess various bioactive functions, but their antiallergic activity remains incompletely defined. Sulfated polysaccharide from Gracilaria lemaneiformis (GLSP) was obtained by water extraction and ethanol precipitation followed by column chromatography. BALB/c mice, RBL-2H3, and KU812 cells were used for verifying the anti food allergic activity of GLSP. According to the results of mice experiment, GLSP was able to alleviate allergy symptoms, to reduce TM-specific IgE and IgG1, to suppress Th2 cell polarization, and to promote the function of regulatory T (Treg) cells. In addition, GLSP had the ability to inhibit the function of RBL-2H3 cells. Furthermore, GLSP inhibited the activation of KU812 via suppression of p38 mitogen-activated protein kinase (MAPK). In conclusion, immunosuppression as well as the reduction in the level of p38 MAPK may contribute to GLSP's putative activity against food allergy. GLSP may be used as a functional food component for allergic patients.

  4. Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways.

    PubMed

    Noh, Eun-Mi; Park, Yeon-Ju; Kim, Jeong-Mi; Kim, Mi-Seong; Kim, Ha-Rim; Song, Hyun-Kyung; Hong, On-Yu; So, Hong-Seob; Yang, Sei-Hoon; Kim, Jong-Suk; Park, Samg Hyun; Youn, Hyun-Jo; You, Yong-Ouk; Choi, Ki-Bang; Kwon, Kang-Beom; Lee, Young-Rae

    2015-10-01

    Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3',4',7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis.

  5. Molecular cloning and expression analysis of mulberry MAPK gene family.

    PubMed

    Wei, Congjin; Liu, Xueqin; Long, Dingpei; Guo, Qing; Fang, Yuan; Bian, Chenkai; Zhang, Dayan; Zeng, Qiwei; Xiang, Zhonghuai; Zhao, Aichun

    2014-04-01

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses in plants. Although MAPKs have been identified and characterized in a few model plants, there is little information available for mulberry Morus sp. L., one of the most ecologically and economically important perennial trees. This study identified 47 mulberry Morus notabilis MAPK (MnMAPK) family genes: 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes based on a genome-wide analysis of the morus genome database. Comparative analysis with MAPK gene families from other plants suggested that MnMAPKs could be divided into five subfamilies (groups A, B, C, D and E) and they could have similar functions in response to abiotic and biotic stresses. MnMAPK gene expression analysis of different stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and methyl jasmonate (MeJA)) revealed that all ten MnMAPK genes responded to high/low temperature, salt and drought stresses, and that nine of the ten MnMAPKs (MnMAPK7 excepted) could be induced by ABA, SA, H2O2 and MeJA, which suggested that MnMAPKs may play pivotal roles in signal transduction pathways. Our results indicated that almost all of the MnMAPKs may be involved in environmental stress and defense responses, which provides the basis for further characterization of the physiological functions of MnMAPKs.

  6. Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1

    PubMed Central

    Zgheel, Faraj; Alhosin, Mahmoud; Rashid, Sherzad; Burban, Mélanie; Auger, Cyril; Schini-Kerth, Valérie B.

    2014-01-01

    Aims Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation. Methods and Results EPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively. Conclusion Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS. PMID:25133540

  7. Baicalein, an active component of Scutellaria baicalensis Georgi, prevents lysophosphatidylcholine-induced cardiac injury by reducing reactive oxygen species production, calcium overload and apoptosis via MAPK pathways

    PubMed Central

    2014-01-01

    Background Lysophosphatidylcholine (lysoPC), a metabolite from membrane phospholipids, accumulates in the ischemic myocardium and plays an important role in the development of myocardial dysfunction ventricular arrhythmia. In this study, we investigated if baicalein, a major component of Huang Qui, can protect against lysoPC-induced cytotoxicity in rat H9c2 embryonic cardiomyocytes. Methods Cell viability was detected by the MTT assay; ROS levels were assessed using DCFH-DA; and intracellular free calcium concentrations were assayed by spectrofluorophotometer. Cell apoptosis and necrosis were evaluated by the flow cytometry assay and Hoechst staining. Mitogen-Activated Protein Kinases (MAPKs), which included the ERK, JNK, and p38, and the apoptotic mechanisms including Bcl-2/Bax, caspase-3, caspase-9 and cytochrome c pathways were examined by Western blot analysis. The activation of MAPKs was examined by enzyme-linked immunosorbent assay. Results We found that lysoPC induced death and apoptosis of H9c2 cells in a dose-dependent manner. Baicalein could prevent lysoPC-induced cell death, production of reactive oxygen species (ROS), and increase of intracellular calcium concentration in H9c2 cardiomyoctes. In addition, baicalein also inhibited lysoPC-induced apoptosis, with associated decreased pro-apoptotic Bax protein, increased anti-apoptotic Bcl-2 protein, resulting in an increase in the Bcl-2/Bax ratio. Finally, baicalein attenuated lysoPC-induced the expression of cytochrome c, casapase-3, casapase-9, and the phosphorylations of ERK1/2, JNK, and p38. LysoPC-induced ERK1/2, JNK, and p38 activations were inhibited by baicalein. Conclusions Baicalein protects cardiomyocytes from lysoPC-induced apoptosis by reducing ROS production, inhibition of calcium overload, and deactivations of MAPK signaling pathways. PMID:25012390

  8. Saikosaponin-D reduces cisplatin-induced nephrotoxicity by repressing ROS-mediated activation of MAPK and NF-κB signalling pathways.

    PubMed

    Ma, Xiaobin; Dang, Chengxue; Kang, Huafeng; Dai, Zhijun; Lin, Shuai; Guan, Haitao; Liu, Xiaoxu; Wang, Xijing; Hui, Wentao

    2015-09-01

    The nephrotoxicity induced by cisplatin (DDP) severely limits the clinical efficacy of this widely used anticancer agent. The observed nephrotoxicity may be the result of DDP-induced inflammation and apoptosis. Saikosaponin-D (SSD), a triterpenoid saponin, has numerous pharmacological properties. The goal of the present study was to investigate whether and how SSD protected against DDP-induced nephrotoxicity. Non-cytotoxic levels of SSD significantly increased the viability rate, improved the nuclear morphology, and attenuated the caspase-3 activation and programmed apoptosis of DDP-treated HK-2 cells. In addition, SSD treatment markedly inhibited the release of tumour necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6), as well as the production of nitric oxide and the expression of inducible nitric oxide synthase (iNOS) by these cells. More importantly, SSD effectively blocked the DDP-induced activation of NF-κB, P38, JNK, and MAPKs. Furthermore, we found that U0126 (a specific inhibitor of MAPKs) strongly inhibited the IKK/IκB/NF-κB-dependent release of pro-inflammatory cytokines and iNOS gene expression. Finally, we demonstrated that SSD decreased the level of reactive oxygen species (ROS) accumulation and that the specific ROS scavenger N-acetylcysteine (NAC) markedly inhibited the DDP-induced activation of MAPK and phosphorylation of the downstream signal NF-κB, which in turn reduced the levels of pro-inflammatory cytokine release and iNOS gene expression. Our results suggest that the SSD-mediated alleviation of DDP-induced nephrotoxicity was due to uncoupling of the ROS, P38, and JNK/NF-κB signalling pathways. PMID:26118633

  9. IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

    PubMed

    Kim, J-H; Song, A-R; Sohn, H-J; Lee, J; Yoo, J-K; Kwon, D; Shin, H-J

    2013-01-01

    Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

  10. Coriolus versicolor mushroom polysaccharides exert immunoregulatory effects on mouse B cells via membrane Ig and TLR-4 to activate the MAPK and NF-κB signaling pathways.

    PubMed

    Yang, Shu-fa; Zhuang, Tai-feng; Si, Yan-mei; Qi, Ke-yan; Zhao, Juan

    2015-03-01

    This study aimed to characterize the immunopotentiating effects and immune receptors for Coriolus versicolor mushroom polysaccharides (CVP), a Chinese medicinal fungus that exerts anti-tumor activities by enhancing host immunity. Proliferation assays were used to determine whether CVP could activate splenocytes. Flow cytometry analysis and IgM and IgG detection were used to characterize CVP-binding cells. Immune receptors were analyzed in immunoprecipitation and western blot assays. The downstream signaling pathways were identified by western blotting or immunostaining. CVP significantly stimulated the proliferation of mouse splenocytes. Fluorescence-labeled CVP (fl-CVP) selectively stained mouse B cells, but not T cells. CVP induced the production of IgM and IgG1 with or without exogenous IL-4. Membrane Ig (B cell antigen-receptor, BCR) was identified as a CVP-binding protein in immunoprecipitation and western blot experiments. CVP-induced B cell proliferation could be significantly inhibited by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ). Phosphorylation of ERK-1/2 and p38 MAPK were clearly increased in a time-dependent manner, as was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP stimulation. Together, we demonstrate that CVP can bind and induce B cell activation using membrane Ig and TLR-4 as potential immune receptors. CVP activates mouse B cells through the MAPK and NF-κB signaling pathways.

  11. Anti-inflammatory activity on mice of extract of Ganoderma lucidum grown on rice via modulation of MAPK and NF-κB pathways.

    PubMed

    Hasnat, Md Abul; Pervin, Mehnaz; Cha, Kyu Min; Kim, Si Kwan; Lim, Beong Ou

    2015-06-01

    Ganoderma lucidum is a popular medicinal mushroom with anti-inflammatory potential. In the present study, the aim was to determine the anti-inflammatory effect and mode of action of G. lucidum grown on germinated brown rice (GLBR) in a mouse model of colitis. It was shown that GLBR suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated macrophages and decreased the expression of COX-2, TNF-α, iNOS, IL-1β, IL-6, and IL-10 mRNAs. GLBR also inhibited activation of p38, ERK, JNK, MAPKs, and nuclear factor kappa-B (NF-κB). In a mouse model of colitis, colonic mucosal injury was evaluated using macroscopic, biochemical, and histopathological testing. Disease activity index (DAI), macroscopic score, and histological score significantly decreased upon GLBR treatment. Moreover, immunofluorescence studies indicated that DSS activates nuclear translocation of NF-κB in colon tissue, which is attenuated by GLBR extract. These findings suggest that GLBR is protective against colitis via inhibition of MAPK phosphorylation and NF-κB activation.

  12. The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells

    PubMed Central

    Ulbrich, Felix; Kaufmann, Kai B.; Meske, Alexander; Lagrèze, Wolf A.; Augustynik, Michael; Buerkle, Hartmut; Ramao, Carlos C.; Biermann, Julia

    2016-01-01

    Purpose Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage. Methods IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections. Results ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects. Conclusion In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI. PMID:27764224

  13. Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-κB and MAPK activation, and acetylation of histone H3.

    PubMed

    Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

    2011-12-01

    Although inflammation acts as host defense mechanism against infection or injury and is primarily a self limiting process, inadequate resolution of inflammatory responses leads to various chronic disorders. This work aimed to elucidate the anti-inflammatory effects of 2-methoxy-4-vinylphenol (2M4VP) isolated from pine needles in LPS-stimulated RAW264.7 cells. Some key pro-inflammatory mediators including nitric oxide (NO), prostaglandins (PGE(2)), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied by sandwich ELISA and western blot. In addition, suppression of NF-κB and MAPK activation, and histone acetylation was studied by western blot analysis and immunostaining. 2M4VP dosedependently inhibited NO and PGE(2) production and also blocked LPS-induced iNOS and COX-2 expression. In addition, 2M4VP potently inhibited the translocation of NF-κB p65 into the nucleus by IκB degradation following IκB-α phosphorylation and the phosphorylation of MAPKs such as p38, ERK1/2, and JNK. Also, 2M4VP inhibited hyper-acetylation of histone H3 (Lys9/Lys14) induced by LPS. Taken together, our results suggest that 2M4VP, a naturally occurring phenolic compound, exert potent anti-inflammatory effects by inhibiting LPS-induced NO, PGE(2), iNOS, and COX-2 in RAW264.7 cells. These effects are mediated by suppression of NF-κB and MAPK activation and histone acetylation.

  14. GENE REGULATION BY MAPK SUBSTRATE COMPETITION

    PubMed Central

    Kim, Yoosik; Andreu, María José; Lim, Bomyi; Chung, Kwanghun; Terayama, Mark; Jiménez, Gerardo; Berg, Celeste A.; Lu, Hang; Shvartsman, Stanislav Y.

    2011-01-01

    SUMMARY Developing tissues are patterned by coordinated activities of signaling systems, which can be integrated by a regulatory region of a gene that binds multiple transcription factors or by a transcription factor that is modified by multiple enzymes. Based on a combination of genetic and imaging experiments in the early Drosophila embryo, we describe a signal integration mechanism that cannot be reduced to a single gene regulatory element or a single transcription factor. This mechanism relies on an enzymatic network formed by Mitogen Activated Protein Kinase (MAPK) and its substrates. Specifically, anteriorly localized MAPK substrates, such as Bicoid, antagonize MAPK-dependent downregulation of Capicua, a repressor which is involved in gene regulation along the dorsoventral axis of the embryo. MAPK substrate competition provides a basis for ternary interaction of the anterior, dorsoventral, and terminal patterning systems. A mathematical model of this interaction can explain gene expression patterns with both anteroposterior and dorsoventral polarities. PMID:21664584

  15. Aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells

    PubMed Central

    Zeng, Yan-Ping; Yang, Chao; Li, Yuan; Fan, Yong; Yang, Hong-Jun; Liu, Bin; Sang, Hong-Xun

    2016-01-01

    Aspirin is a commonly used medicine as an effective antipyretic, analgesic and anti-inflammatory drug. Previous studies have demonstrated its potential effects of anti-postmenopausal osteoporosis, while the molecular mechanisms remain unclear. The effects of aspirin on receptor-activator of nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclasts were investigated in RAW264.7 cells in the current study. Using tartrate-resistant acid phosphatase (TRAP) staining, it was observed that aspirin inhibited the differentiation of RANKL-induced RAW264.7 cells. The mRNA expression of osteoclastic marker genes, including cathepsin K, TRAP, matrix metalloproteinase 9 and calcitonin receptor, were suppressed by aspirin as identified using reverse transcription-quantitative polymerase chain reaction analysis. The immunofluorescence assay indicated that aspirin markedly inhibited NF-κB p65 translocation to the nucleus in RANKL-induced RAW264.7 cells. In addition, aspirin also suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), observed by western blot analysis. Taken together, these data identified that aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells, implying that aspirin may possess therapeutic potential for use in the prevention and treatment of osteoporosis. PMID:27430169

  16. Activation of MAPK pathways due to DUSP4 loss promotes cancer stem cell-like phenotypes in basal-like breast cancer.

    PubMed

    Balko, Justin M; Schwarz, Luis J; Bhola, Neil E; Kurupi, Richard; Owens, Phillip; Miller, Todd W; Gómez, Henry; Cook, Rebecca S; Arteaga, Carlos L

    2013-10-15

    Basal-like breast cancer (BLBC) is an aggressive disease that lacks a clinically approved targeted therapy. Traditional chemotherapy is effective in BLBC, but it spares the cancer stem cell (CSC)-like population, which is likely to contribute to cancer recurrence after the initial treatment. Dual specificity phosphatase-4 (DUSP4) is a negative regulator of the mitogen-activated protein kinase (MAPK) pathway that is deficient in highly aggressive BLBCs treated with chemotherapy, leading to aberrant MAPK activation and resistance to taxane-induced apoptosis. Herein, we investigated how DUSP4 regulates the MAP-ERK kinase (MEK) and c-jun-NH2-kinase (JNK) pathways in modifying CSC-like behavior. DUSP4 loss increased mammosphere formation and the expression of the CSC-promoting cytokines interleukin (IL)-6 and IL-8. These effects were caused in part by loss of control of the MEK and JNK pathways and involved downstream activation of the ETS-1 and c-JUN transcription factors. Enforced expression of DUSP4 reduced the CD44(+)/CD24(-) population in multiple BLBC cell lines in a MEK-dependent manner, limiting tumor formation of claudin-low SUM159PT cells in mice. Our findings support the evaluation of MEK and JNK pathway inhibitors as therapeutic agents in BLBC to eliminate the CSC population.

  17. Aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells.

    PubMed

    Zeng, Yan-Ping; Yang, Chao; Li, Yuan; Fan, Yong; Yang, Hong-Jun; Liu, Bin; Sang, Hong-Xun

    2016-09-01

    Aspirin is a commonly used medicine as an effective antipyretic, analgesic and anti-inflammatory drug. Previous studies have demonstrated its potential effects of anti-postmenopausal osteoporosis, while the molecular mechanisms remain unclear. The effects of aspirin on receptor‑activator of nuclear factor κB (NF‑κB) ligand (RANKL)‑induced osteoclasts were investigated in RAW264.7 cells in the current study. Using tartrate‑resistant acid phosphatase (TRAP) staining, it was observed that aspirin inhibited the differentiation of RANKL‑induced RAW264.7 cells. The mRNA expression of osteoclastic marker genes, including cathepsin K, TRAP, matrix metalloproteinase 9 and calcitonin receptor, were suppressed by aspirin as identified using reverse transcription‑quantitative polymerase chain reaction analysis. The immunofluorescence assay indicated that aspirin markedly inhibited NF‑κB p65 translocation to the nucleus in RANKL‑induced RAW264.7 cells. In addition, aspirin also suppressed the phosphorylation of mitogen‑activated protein kinases (MAPKs), observed by western blot analysis. Taken together, these data identified that aspirin inhibits osteoclastogenesis by suppressing the activation of NF‑κB and MAPKs in RANKL‑induced RAW264.7 cells, implying that aspirin may possess therapeutic potential for use in the prevention and treatment of osteoporosis. PMID:27430169

  18. Vesicular Stomatitis Virus Matrix Protein Impairs CD1d-Mediated Antigen Presentation through Activation of the p38 MAPK Pathway▿

    PubMed Central

    Renukaradhya, Gourapura J.; Khan, Masood A.; Shaji, Daniel; Brutkiewicz, Randy R.

    2008-01-01

    Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murine CD1d-mediated antigen presentation to NKT cells. In the present study, it was found that the VSV matrix (VSV-M) protein is an important element in this decrease in antigen presentation postinfection. The VSV-M protein altered the intracellular distribution of murine CD1d molecules, resulting in qualitative (but not quantitative) changes in cell surface CD1d expression. The M protein was distributed throughout the infected cell, and it was found to activate the mitogen-activated protein kinase (MAPK) p38 very early postinfection. Infection of CD1d+ cells with a temperature-sensitive VSV-M mutant at the nonpermissive temperature both substantially reversed the inhibition of antigen presentation by CD1d and delayed the activation of p38. Thus, the VSV-M protein plays an important role in permitting the virus to evade important components of the innate immune response by regulating specific MAPK pathways. PMID:18815300

  19. Exogenous hydrogen sulfide promotes C6 glioma cell growth through activation of the p38 MAPK/ERK1/2-COX-2 pathways.

    PubMed

    Zhen, Yulan; Zhang, Wei; Liu, Chujie; He, Jing; Lu, Yun; Guo, Ruixian; Feng, Jianqiang; Zhang, Ying; Chen, Jingfu

    2015-11-01

    Hydrogen sulfide (H2S) participates in multifarious physiological and pathophysiologic progresses of cancer both in vitro and in vivo. We have previously demonstrated that exogenous H2S promoted liver cancer cells proliferation/anti‑apoptosis/angiogenesis/migration effects via amplifying the activation of NF-κB pathway. However, the effects of H2S on cancer cell proliferation and apoptosis are controversial and remain unclear in C6 glioma cells. The present study investigated the effects of exogenous H2S on cancer cells growth via activating p38 MAPK/ERK1/2-COX-2 pathways in C6 glioma cells. C6 glioma cells were treated with 400 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated (p)-p38 MAPK, total (t)-p38 MAPK, p-ERK1/2, t-ERK1/2, cyclooxygenase-2 (COX-2) and caspase-3 were measured by western blotting assay. Cell viability was detected by Cell Counting Kit-8 (CCK-8). Apoptotic cells were observed by Hoechst 33258 staining assay. Cell proliferation was directly detected under fully automatic inverted microscope. Exposure of C6 glioma cells to NaHS resulted in cell proliferation, as evidenced by an increase in cell viability. In addition, NaHS treatment reduced apoptosis, as indicated by the decreased apoptotic percentage and the cleaved caspase-3 expression. Importantly, exposure of the cells to NaHS increased the expression levels of p-p38 MAPK, p-ERK1/2 and COX-2. Notably, co-treatment of C6 glioma cells with 400 µmol/l NaHS and AOAA (an inhibitor of CBS) largely suppressed the above NaHS-induced effects. Combined treatment with NaHS and SB203580 (an inhibitor of p38 MAPK) or PD-98059 (an inhibitor of ERK1/2) resulted in the synergistic reduction of COX-2 expression and increase of caspase-3 expression, a decreased number of apoptotic cells, along with decreased cell viability. Combined treatment with NS-398 (an inhibitor of COX-2) and NaHS also resulted in the synergistic increase of caspase-3, a decreased in the

  20. Thymol has antifungal activity against Candida albicans during infection and maintains the innate immune response required for function of the p38 MAPK signaling pathway in Caenorhabditis elegans.

    PubMed

    Shu, Chengjie; Sun, Lingmei; Zhang, Weiming

    2016-08-01

    The Caenorhabditis elegans model can be used to study Candida albicans virulence and host immunity, as well as to identify plant-derived natural products to use against C. albicans. Thymol is a hydrophobic phenol compound from the aromatic plant thyme. In this study, the in vitro data demonstrated concentration-dependent thymol inhibition of both C. albicans growth and biofilm formation during different developmental phases. With the aid of the C. elegans system, we performed in vivo assays, and our results further showed the ability of thymol to increase C. elegans life span during infection, inhibit C. albicans colony formation in the C. elegans intestine, and increase the expression levels of host antimicrobial genes. Moreover, among the genes that encode the p38 MAPK signaling pathway, mutation of the pmk-1 or sek-1 gene decreased the beneficial effects of thymol's antifungal activity against C. albicans and thymol's maintenance of the innate immune response in nematodes. Western blot data showed the level of phosphorylation of pmk-1 was dramatically decreased against C. albicans. In nematodes, treatment with thymol recovered the dysregulation of pmk-1 and sek-1 gene expressions, the phosphorylation level of PMK-1 caused by C. albicans infection. Therefore, thymol may act, at least in part, through the function of the p38 MAPK signaling pathway to protect against C. albicans infection and maintain the host innate immune response to C. albicans. Our results indicate that the p38 MAPK signaling pathway plays a crucial role in regulating the beneficial effects observed after nematodes infected with C. albicans were treated with thymol. PMID:26783030

  1. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-{kappa}B and MAPK pathways

    SciTech Connect

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. Black-Right-Pointing-Pointer Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. Black-Right-Pointing-Pointer The mode of action of this limonoid is by inhibiting activation of the NF-{kappa}B and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-{kappa}B (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 {mu}M. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-{kappa}B p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

  2. [Insulin combined with selenium inhibit p38MAPK/CBP pathway and suppresses cardiomyocyte apoptosis in rats with diabetic cardiomyopathy].

    PubMed

    Xu, Tianjiao; Liu, Yong; Deng, Yating; Meng, Jin; Li, Ping; Xu, Xiaoli; Zeng, Jurong

    2016-07-01

    Objective To investigate the effect of insulin in combination with selenium on p38-mitogen-activated protein kinase/CREB-binding protein (p38MAPK/CBP) pathway in rats with diabetic cardiomyopathy. Methods Fifty SD rats were randomly grouped into control group, diabetic cardiomyopathy (DCM) group, diabetic cardiomyopathy with insulin treatment (DCM-In) group, diabetic cardiomyopathy with selenium treatment (DCM-Se) group, and diabetic cardiomyopathy with insulin and selenium combination treatment (DCM-In-Se) group. Flow cytometry was used to analyze cell cycle. TUNEL staining was used to detect cardiomyocyte apoptosis. Western blotting was used to examine the levels of cyclin D1, cyclin E, Bax, Bcl-2, p38MAPK, p-p38MAPK, CBP and Ku70. Co-immunoprecipitation was used to examine the acetylation status of Ku70. Results Insulin in combination with selenium significantly inhibited cardiomyocyte apoptosis, increased Bcl-2 levels and decreased Bax, cyclin D1, cyclin E, p38MAPK, p-p38MAPK, CBP, Ku70 and acetylated Ku70 levels. Conclusion The combined treatment of insulin and selenium suppresses cardiomyocyte apoptosis by inhibiting p38MAPK/CBP pathway. PMID:27363274

  3. Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation

    PubMed Central

    Zhang, Xiyu; Wang, Xiyao; Wu, Tingting; Li, Boxuan; Liu, Tianqi; Wang, Rong; Liu, Qiao; Liu, Zhaojian; Gong, Yaoqin; Shao, Changshun

    2015-01-01

    Isoliensinine, liensinine and neferine are major bisbenzylisoquinoline alkaloids in the seed embryo of lotus (Nelumbo nucifera), and exhibit potential anti-cancer activity. Here, we explored the effects of these alkaloids on triple-negative breast cancer cells and found that among the three alkaloids isoliensinine possesses the most potent cytotoxic effect, primarily by inducing apoptosis. Interestingly, isoliensinine showed a much lower cytotoxicity against MCF-10A, a normal human breast epithelial cell line. Further studies showed that isoliensinine could significantly increase the production of reactive oxygen species (ROS) in triple-negative breast cancer cells, but not in MCF-10A cells. The isoliensinine-induced apoptosis could be attenuated by radical oxygen scavenger N-acetyl cysteine, suggesting that the cytotoxic effect of isoliensinine on cancer cells is at least partially achieved by inducing oxidative stress. We found that both p38 MAPK and JNK signaling pathways were activated by isoliensinine treatment and contributed to the induction of apoptosis. Furthermore, inhibitors or specific siRNAs of p38 MAPK and JNK could attenuate apoptosis induced by isoliensinine. However, only the p38 inhibitor or p38-specific siRNA blocked the elevation of ROS in isoliensinine-treated cells. Our findings thus revealed a novel antitumor effect of isoliensinine on breast cancer cells and may have therapeutic implications. PMID:26219228

  4. Matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro.

    PubMed

    Lu, Shijin; Xiao, Xungang; Cheng, Minghua

    2015-01-01

    Interleukin (IL)-1β plays an important role in promoting osteoarthritis (OA) lesions by inducing chondrocytes to secrete matrix metalloproteinases (MMPs), which degrade the extracellular matrix and facilitate chondrocyte apoptosis. Matrine was shown to exert anti-inflammatory effects. However, the role of matrine in OA is still unclear. Therefore, in this study, we investigated the effects of matrine on the expression of MMPs in IL-1β-treated human chondrocytes and the underlying mechanism. The cell viability of chondrocytes was detected by MTT assay. The cell apoptosis of chondrocytes was measured by flow cytometric analysis. The protein production of MMPs was determined by ELISA. The protein expression of phosphorylation of mitogen-activated protein kinases (MAPKs) and the inhibitor of kappaB alpha (IκBα) was determined by Western blot. Matrine significantly inhibited the IL-1β-induced apoptosis in chondrocytes. It also significantly inhibited the IL-1β-induced release of MMP-3 and MMP-13, and increased the production of TIMP-1. Furthermore, matrine inhibits the phosphorylation of p-38, extracellular regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and IκBα degradation induced by IL-1β in chondrocytes. Taken together, our results show that matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro. Therefore,-matrine may be beneficial in the treatment of OA.

  5. Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation.

    PubMed

    Zhang, Xiyu; Wang, Xiyao; Wu, Tingting; Li, Boxuan; Liu, Tianqi; Wang, Rong; Liu, Qiao; Liu, Zhaojian; Gong, Yaoqin; Shao, Changshun

    2015-01-01

    Isoliensinine, liensinine and neferine are major bisbenzylisoquinoline alkaloids in the seed embryo of lotus (Nelumbo nucifera), and exhibit potential anti-cancer activity. Here, we explored the effects of these alkaloids on triple-negative breast cancer cells and found that among the three alkaloids isoliensinine possesses the most potent cytotoxic effect, primarily by inducing apoptosis. Interestingly, isoliensinine showed a much lower cytotoxicity against MCF-10A, a normal human breast epithelial cell line. Further studies showed that isoliensinine could significantly increase the production of reactive oxygen species (ROS) in triple-negative breast cancer cells, but not in MCF-10A cells. The isoliensinine-induced apoptosis could be attenuated by radical oxygen scavenger N-acetyl cysteine, suggesting that the cytotoxic effect of isoliensinine on cancer cells is at least partially achieved by inducing oxidative stress. We found that both p38 MAPK and JNK signaling pathways were activated by isoliensinine treatment and contributed to the induction of apoptosis. Furthermore, inhibitors or specific siRNAs of p38 MAPK and JNK could attenuate apoptosis induced by isoliensinine. However, only the p38 inhibitor or p38-specific siRNA blocked the elevation of ROS in isoliensinine-treated cells. Our findings thus revealed a novel antitumor effect of isoliensinine on breast cancer cells and may have therapeutic implications. PMID:26219228

  6. Low amino acids affect expression of 11β-HSD2 in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.

    PubMed

    Shang, Yueli; Yang, Xiaojing; Zhang, Rui; Zou, Huafeng; Zhao, Ruqian

    2012-05-01

    Maternal protein restriction diminishes placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11β-HSD2 expression and activity. Despite a significant up-regulation of 11β-HSD2 mRNA expression in LAA cells, 11β-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P<0.05), indicating a mechanism of post-transcriptional regulation. Among 5 miRNAs targeting 11β-HSD2, miR-498 was expressed significantly higher in LAA cells. Leptin concentration was significantly lower (P<0.01) in LAA medium. The mRNA expression of both isoforms of leptin receptor was significantly higher in LAA cells, although no difference was detected at protein level. To further clarify whether leptin is involved in mediating the effect of LAA on 11β-HSD2 activity, leptin was supplemented to LAA medium, whereas three specific inhibitors of leptin signaling pathways, WP1066 for JAK-STAT, PD98059 for MAPK and LY294002 for PI3K, respectively were added to control medium. Leptin restored the diminished 11β-HSD2 activity in LAA cells, whereas WP1066 (5 nM) and PD98059 (50 nM) significantly decreased 11β-HSD2 activity in control cells. In conclusion, the present results indicate that LAA diminishes 11β-HSD2 expression and activity in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.

  7. Cross-talk between p(38)MAPK and G iα in regulating cPLA 2 activity by ET-1 in pulmonary smooth muscle cells.

    PubMed

    Chakraborti, Sajal; Chowdhury, Animesh; Chakraborti, Tapati

    2015-02-01

    Endothelin-1 (ET-1) is known as the most potent vasoconstrictor yet described. Infusion of ET-1 into isolated rabbit lung has been shown to cause pulmonary vasoconstriction with the involvement of arachidonic acid metabolites. Given the potency of arachidonic acid metabolites, the activity of phospholipase A2 must be tightly regulated. Herein, we determined the mechanisms by which ET-1 stimulates cPLA2 activity during ET-1 stimulation of bovine pulmonary artery smooth muscle cells. We demonstrated that (i) treatment of bovine pulmonary artery smooth muscle cells with ET-1 stimulates cPLA2 activity in the cell membrane; (ii) ET-1 caused increase in O 2 (·-) production occurs via NADPH oxidase-dependent mechanism; (iii) ET-1-stimulated NADPH oxidase activity is markedly prevented upon pretreatment with PKC-ζ inhibitor, indicating that PKC-ζ plays a prominent role in this scenario; (iv) ET-1-induced NADPH oxidase-derived O 2 (·-) stimulates an aprotinin sensitive protease activity due to prominent increase in [Ca(2+)]i; (v) the aprotinin sensitive protease plays a pivotal role in activating PKC-α, which in turn phosphorylates p(38)MAPK and subsequently Giα leading to the activation of cPLA2. Taken together, we suggest that cross-talk between p(38)MAPK and Giα with the involvement of PKC-ζ, NADPH oxidase-derived O 2 (·-) , [Ca(2+)]i, aprotinin-sensitive protease and PKC-α play a pivotal role for full activation of cPLA2 during ET-1 stimulation of pulmonary artery smooth muscle cells.

  8. Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

    PubMed Central

    Copik, Alicja. J.; Baldys, Aleksander; Nguyen, Khanh; Sahdeo, Sunil; Ho, Hoangdung; Kosaka, Alan; Dietrich, Paul J.; Fitch, Bill; Raymond, John R.; Ford, Anthony P. D. W.; Button, Donald; Milla, Marcos E.

    2015-01-01

    The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK

  9. TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

    PubMed Central

    Walsh, Matthew C.; Kim, Gregory K.; Maurizio, Paul L.; Molnar, Elizabeth E.; Choi, Yongwon

    2008-01-01

    The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFκB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFκB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFκB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKγ/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFκB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function. PMID:19112497

  10. Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats

    PubMed Central

    Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

    2016-01-01

    Objectives This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. Methods FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Results Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Conclusions Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to

  11. 24-Week Exposure to Oxidized Tyrosine Induces Hepatic Fibrosis Involving Activation of the MAPK/TGF-β1 Signaling Pathway in Sprague-Dawley Rats Model

    PubMed Central

    Li, Zhuqing Leslie; Shi, Yonghui; Le, Guowei; Ding, Yinyi; Zhao, Qi

    2016-01-01

    Scope. Oxidized tyrosine (O-Tyr) has been widely detected in many consumer protein products. O-Tyr products such as dityrosine (Dityr) and 3-nitrotyrosine (3-NT) are universal biomarkers of protein oxidation and have been demonstrated to be associated with metabolic disorders in biological system. Evaluation of potential intracorporal effects of dietary O-Tyr is important since the mechanism of biological impacts induced by oral oxidized protein products (OPPs) is still limited although we have proved that some dietary OPPs would induce oxidative injury to liver and kidney. Methods and Results. The present study aimed to investigate the dose-dependent hepatic injury caused by oral O-Tyr in rats. 24-week feeding of O-Tyr enhanced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, increased total bilirubin (TBiL) content, and led to oxidative damage in rats liver. Besides, O-Tyr distinctly increased the phosphorylation of p38 and ERK2 MAPKs and enhanced fibrosis-related TGF-β1 and Smad2/3 levels. Higher extracellular matrix (ECM) indexes (ICTP, PIIINP) and histological examination (HE and Masson staining) also supported dose-dependent hepatic fibrosis caused by O-Tyr. Conclusion. These findings reveal that O-Tyr may induce oxidative damage and hepatic fibrosis via MAPK/TGF-β1 signaling pathway, in which ROS together with malondialdehyde (MDA) and OPPs act as the pivotal mediators. PMID:26788244

  12. Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network

    PubMed Central

    Springler, Alexandra; Hessenberger, Sabine; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    Deoxynivalenol (DON), produced by the plant pathogens Fusarium graminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways. PMID:27618100

  13. Inhibition of Advanced Glycation Endproduct Formation by Lotus Seedpod Oligomeric Procyanidins through RAGE-MAPK Signaling and NF-κB Activation in High-Fat-Diet Rats.

    PubMed

    Wu, Qian; Li, Shuyi; Li, Xiaopeng; Sui, Yong; Yang, Yan; Dong, Lihong; Xie, Bijun; Sun, Zhida

    2015-08-12

    This study investigated the protective properties of lotus seedpod oligomeric procyanidins (LSOPC) against nonalcoholic fatty liver disease (NAFLD) and its underlying mechanism. Sprague-Dawley (SD) male rats were fed a basic diet, a high-fat diet (HFD), or HFD plus 0.2 or 0.5% (w/w) LSOPC for 12 weeks. Administration of LSOPC markedly reduced serum and hepatic biochemical parameters and protein expression of advanced glycation endproducts (AGEs). Additionally, 0.5% (w/w) LSOPC treatment remarkably reversed the increasing tendency of receptor of advanced glycation endproduct (RAGE) to normal level. Furthermore, dietary LSOPC significantly decreased the protein levels of mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) and down-regulated genes involved in pro-inflammatory cytokines and adhesion molecules. Taken together, these findings demonstrate that LSOPC may protect obese rats with long-term HFD-induced NAFLD against RAGE-MAPK-NF-κB signaling suppression.

  14. Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network.

    PubMed

    Springler, Alexandra; Hessenberger, Sabine; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    Deoxynivalenol (DON), produced by the plant pathogens Fusarium graminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways. PMID:27618100

  15. Protective activity of salidroside against ethanol-induced gastric ulcer via the MAPK/NF-κB pathway in vivo and in vitro.

    PubMed

    Chang, Xiayun; Luo, Fen; Jiang, Wenjiao; Zhu, Lingpeng; Gao, Jin; He, He; Wei, Tingting; Gong, Shilin; Yan, Tianhua

    2015-09-01

    Salidroside (Sal) is a traditional Chinese medicine with various pharmacological effects. The present study aimed to investigate the protective effect of Sal on ethanol-induced acute gastric ulcer and H2O2-induced gastric epithelial cell damage. 0.2 ml ethanol and 400 μM H2O2 were applied to establish a gastric ulcer model in vivo and in vitro respectively. The production of interleukin (IL)-6, interleukin (IL)-1β and tumor necrosis factor (TNF)-α was analyzed, as well as myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD). MTT assay was used to detect cell viability. In addition, MAPK/NF-κB signal pathway-related proteins p-ERK, p-JNK, p-p38, p-IκBα and p-NF-κBp65 were analyzed to determine the underlying protective mechanism. Downstream genes such as cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and leukotrienes B4 (LTB4) were also measured. Obtained data indicated that Sal inhibited the overproduction of pro-inflammatory cytokines and enhanced antioxidant activity. Collectively, it is assumed that Sal could alleviate ethanol-induced acute gastric ulcer and H2O2-induced gastric epithelial cell damage through the MAPK/NF-κB pathway.

  16. Sulforaphane Ameliorates 3-Nitropropionic Acid-Induced Striatal Toxicity by Activating the Keap1-Nrf2-ARE Pathway and Inhibiting the MAPKs and NF-κB Pathways.

    PubMed

    Jang, Minhee; Cho, Ik-Hyun

    2016-05-01

    The potential neuroprotective value of sulforaphane (SFN) in Huntington's disease (HD) has not been established yet. We investigated whether SFN prevents and improves the neurological impairment and striatal cell death in a 3-nitropropionic acid (3-NP)-induced mouse model of HD. SFN (2.5 and 5.0 mg/kg/day, i.p.) was given daily 30 min before 3-NP treatment (pretreatment) and from onset/progression/peak points of the neurological scores. Pretreatment with SFN (5.0 mg/kg/day) produced the best neuroprotective effect with respect to the neurological scores and lethality among other conditions. The protective effects due to pretreatment with SFN were associated with the following: suppression of the formation of a lesion area, neuronal death, succinate dehydrogenase activity, apoptosis, microglial activation, and mRNA or protein expression of inflammatory mediators, including tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase, and cyclooxygenase-2 in the striatum after 3-NP treatment. Also, pretreatment with SFN activated the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and inhibited the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways in the striatum after 3-NP treatment. As expected, the pretreatment with activators (dimethyl fumarate and antioxidant response element inducer-3) of the Keap1-Nrf2-ARE pathway decreased the neurological impairment and lethality after 3-NP treatment. Our findings suggest that SFN may effectively attenuate 3-NP-induced striatal toxicity by activating the Keap1-Nrf2-ARE pathway and inhibiting the MAPKs and NF-κB pathways and that SFN has a wide therapeutic time-window for HD-like symptoms. PMID:26096705

  17. Activation of p44/42 MAPK Plays a Role in the TBT-induced Loss of Human Natural Killer (NK) Cell Function

    PubMed Central

    Dudimah, Fred D.; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M.

    2009-01-01

    Natural Killer (NK) cells destroy (lyse) tumor cells, virally infected cells and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as Phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function (51Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1 h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1 h exposure to 5 nM PMA caused a 6 fold increase in phospho-p44/42 levels. Previous studies showed a 5 fold increase in phospho-p44/42 in response to a 1 h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function. PMID:20213532

  18. Cadmium at nanomolar concentrations activates Raf-MEK-ERK1/2 MAPKs signaling via EGFR in human cancer cell lines.

    PubMed

    Ali, Imran; Damdimopoulou, Pauliina; Stenius, Ulla; Halldin, Krister

    2015-04-25

    Cadmium (Cd) is an environmental contaminant classified as carcinogenic to humans by the International Agency for Research on Cancer, supported by data from occupational exposure. Environmentally relevant dietary exposure to Cd has recently been associated with osteoporosis and cancers of the prostate, endometrium, and breast in the general population. The low exposure effects have been proposed to result from endocrine modulative properties of Cd, which mimic the physiological actions of estrogen and androgen. However, the mechanism of action of Cd is an unanswered question. We have shown previously, using mouse models, that canonical estrogen receptor signaling is not involved in estrogen mimicry effects of Cd. Instead, low-level Cd exposure stimulated the mitogen-activated protein kinases (MAPKs) ERK1/2 in these mice. Here we investigate further the ERK1/2 MAPK signaling activation by Cd in vitro by using nanomolar concentrations of cadmium chloride (CdCl2) in three different human carcinoma cell lines: HepG2, MCF-7, and ECC-1. The findings also were confirmed in previously collected mouse tissue samples. We show that 10(-8)M levels of CdCl2 activate ERK1/2 (Tyr 204) and the p53 specific ubiquitin ligase Mdm2 (Ser 166) via Raf and MEK by acting through the epidermal growth factor receptor (EGFR). Furthermore, our results suggest that the CdCl2-induced activation of ERK1/2 and Mdm2 may interfere with the p53 response to genotoxic compounds in cancer cell lines. Our data collectively suggest that nanomolar levels of CdCl2 activate Raf-MEK-ERK1/2 via EGFR. We hypothesize that this signaling cascade may be involved in observed low exposure effects of Cd in certain human populations.

  19. The procyanidin trimer C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes.

    PubMed

    Sung, Nak-Yun; Yang, Mi-So; Song, Du-Sup; Byun, Eui-Baek; Kim, Jae-Kyung; Park, Jong-Heum; Song, Beom-Seok; Lee, Ju-Woon; Park, Sang-Hyun; Park, Hyun-Jin; Byun, Myung-Woo; Byun, Eui-Hong; Kim, Jae-Hun

    2013-08-15

    Numerous studies have shown various relationships between foods with a high nutritional value and a robust immune response, particularly studies that have focused on host protection and cytokine networks. This study aimed to clarify the role played by the procyanidin trimer C1 in innate and adaptive immunity. Procyanidin C1 did not exert cytotoxicity at concentrations ranging from 7.8 to 62.5 μg/ml in macrophage cells; therefore, concentration of 62.5 μg/ml was used as the maximum dose of procyanidin C1 throughout subsequent experiments. Procyanidin C1 enhanced inducible nitric oxide synthase-mediated nitric oxide production in a concentration-dependent manner. In addition, procyanidin C1 functionally induced macrophage activation by augmenting the expression of cell surface molecules (CD80, CD86, and MHC II) and proinflammatory cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) via activation of mitogen-activated protein kinase (MAPK), e.g., p38, ERK, and JNK and nuclear factor (NF)-κB signaling pathways. Interestingly, procyanidin C1 effectively polarized T helper type 1 (Th1) by secreting Th1-mediated cytokines (interferon-γ, IL-12p70, and IL-2) and inducing splenocyte proliferation, indicating that procyanidin C1 contributes to Th1 polarization of the immune response. Accordingly, these findings confirms that the procyanidin C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes, which suggests that procyanidin C1 regulates innate and adaptive immunity by macrophage activation and Th1 polarization.

  20. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation.

    PubMed

    Lochhead, Pamela A; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R; Wedge, Stephen R; Cook, Simon J

    2016-01-01

    ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

  1. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

    PubMed Central

    Lochhead, Pamela A.; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R.; Wedge, Stephen R.; Cook, Simon J.

    2016-01-01

    Abstract ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRASG12C/G13D or BRAFV600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target. PMID:26959608

  2. Prodigiosin isolated from Hahella chejuensis suppresses lipopolysaccharide-induced NO production by inhibiting p38 MAPK, JNK and NF-kappaB activation in murine peritoneal macrophages.

    PubMed

    Huh, Jung-Eun; Yim, Joung-Han; Lee, Hong-Kum; Moon, Eun-Yi; Rhee, Dong-Kwon; Pyo, Suhkneung

    2007-12-15

    Prodigiosin was isolated from marine bacteria Hahella chejuensis which has been recently discovered from Marado, Cheju Island, Republic of Korea. Immunosuppressive properties have been reported for prodigiosin members such as undecylprodigiosin, metacycloprodigiosin, prodigiosin and its synthetic analogue PNU156804 (PNU). However, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of prodigiosin on the production of inflammatory cytokines and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine macrophage. When thioglycollate-elicited macrophages pre-exposed to prodigiosin (1-100 ng/ml) were stimulated with LPS, pretreatment with prodigiosin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In contrast, the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 was not altered. Inhibition of iNOS protein expression appears to be at the transcriptional level, since prodigiosin decreased LPS-induced NF-kappaB activity through preventing the degradation of IkBalpha, with significant inhibition achieved following pretreatment with prodigiosin. However, prodigiosin did not exert any effect on AP-1 activity. Prodigiosin blocked phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK), but not that of extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of NO in macrophages. Taken together, the present data suggest that prodigiosin reduces NO production and iNOS expression by inhibiting LPS-triggered p38 MAPK and JNK phosphorylation and NF-kappaB activation, thereby implicating a mechanism by which prodigiosin may exert its immunosuppressive effects.

  3. Online social activity reflects economic status

    NASA Astrophysics Data System (ADS)

    Liu, Jin-Hu; Wang, Jun; Shao, Junming; Zhou, Tao

    2016-09-01

    To characterize economic development and diagnose the economic health condition, several popular indices such as gross domestic product (GDP), industrial structure and income growth are widely applied. However, computing these indices based on traditional economic census is usually costly and resources consuming, and more importantly, following a long time delay. In this paper, we analyzed nearly 200 million users' activities for four consecutive years in the largest social network (Sina Microblog) in China, aiming at exploring latent relationships between the online social activities and local economic status. Results indicate that online social activity has a strong correlation with local economic development and industrial structure, and more interestingly, allows revealing the macro-economic structure instantaneously with nearly no cost. Beyond, this work also provides a new venue to identify risky signal in local economic structure.

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

    PubMed Central

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

    2015-01-01

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

  5. Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors

    SciTech Connect

    Jorda, Mireia; Vinyals, Antonia; Marazuela, Anna; Cubillo, Eva; Olmeda, David; Valero, Eva; Cano, Amparo; Fabra, Angels . E-mail: afabra@idibell.org

    2007-07-01

    Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors.

  6. The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

    PubMed Central

    Colombatti, Marco; Fracasso, Giulio; Scupoli, Maria Teresa; Cingarlini, Sara; Poffe, Ornella; Naim, Hassan Y.; Heine, Martin; Tridente, Giuseppe; Mainiero, Fabrizio; Ramarli, Dunia

    2009-01-01

    The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis. PMID:19242540

  7. Silica nanoparticles induce oxidative stress, inflammation, and endothelial dysfunction in vitro via activation of the MAPK/Nrf2 pathway and nuclear factor-κB signaling

    PubMed Central

    Guo, Caixia; Xia, Yinye; Niu, Piye; Jiang, Lizhen; Duan, Junchao; Yu, Yang; Zhou, Xianqing; Li, Yanbo; Sun, Zhiwei

    2015-01-01

    Despite the widespread application of silica nanoparticles (SiNPs) in industrial, commercial, and biomedical fields, their response to human cells has not been fully elucidated. Overall, little is known about the toxicological effects of SiNPs on the cardiovascular system. In this study, SiNPs with a 58 nm diameter were used to study their interaction with human umbilical vein endothelial cells (HUVECs). Dose- and time-dependent decrease in cell viability and damage on cell plasma-membrane integrity showed the cytotoxic potential of the SiNPs. SiNPs were found to induce oxidative stress, as evidenced by the significant elevation of reactive oxygen species generation and malondialdehyde production and downregulated activity in glutathione peroxidase. SiNPs also stimulated release of cytoprotective nitric oxide (NO) and upregulated inducible nitric oxide synthase (NOS) messenger ribonucleic acid, while downregulating endothelial NOS and ET-1 messenger ribonucleic acid, suggesting that SiNPs disturbed the NO/NOS system. SiNP-induced oxidative stress and NO/NOS imbalance resulted in endothelial dysfunction. SiNPs induced inflammation characterized by the upregulation of key inflammatory mediators, including IL-1β, IL-6, IL-8, TNFα, ICAM-1, VCAM-1, and MCP-1. In addition, SiNPs triggered the activation of the Nrf2-mediated antioxidant system, as evidenced by the induction of nuclear factor-κB and MAPK pathway activation. Our findings demonstrated that SiNPs could induce oxidative stress, inflammation, and NO/NOS system imbalance, and eventually lead to endothelial dysfunction via activation of the MAPK/Nrf2 pathway and nuclear factor-κB signaling. This study indicated a potential deleterious effect of SiNPs on the vascular endothelium, which warrants more careful assessment of SiNPs before their application. PMID:25759575

  8. Upregulation of contractile endothelin type B receptors by lipid-soluble cigarette smoking particles in rat cerebral arteries via activation of MAPK

    SciTech Connect

    Sandhu, Hardip; Xu, Cang Bao; Edvinsson, Lars

    2010-11-15

    Cigarette smoke exposure increases the risk of stroke. However, the underlying molecular mechanisms are poorly understood. Endothelin system plays key roles in the pathogenesis of stroke. The present study was designed to examine if lipid-soluble (dimethyl sulfoxide-soluble) cigarette smoke particles (DSP) induces upregulation of contractile endothelin type B (ET{sub B}) receptors in rat cerebral arteries and if activation of mitogen activated protein kinase (MAPK) and nuclear factor-kappaB (NF-{kappa}B) mediate the upregulation of contractile endothelin receptors in the cerebral arteries. Rat middle cerebral arteries were isolated and organ cultured in serum free medium for 24 h in the presence of DSP with or without specific inhibitors: MEK specific (U0126), p38 specific (SB202190), JNK specific (SP600125), NF-{kappa}B specific (BMS-345541) or (IMD-0354), transcription inhibitor (actinomycin D), or translation blocker (cycloheximide). Contractile responses to the ET{sub B} receptor agonist sarafotoxin 6c were investigated by a sensitive myograph. The expression of the ET{sub B} receptors were studied at mRNA and protein levels using quantitative real time PCR and immunohistochemistry, respectively. Results show that organ culture per se induced transcriptional upregulation of contractile ET{sub B} receptors in the cerebral vascular smooth muscle cells. This upregulation was further increased at the translational level by addition of DSP to the organ culture, but this increase was not seen by addition of nicotine or water-soluble cigarette smoke particles to the organ culture. The increased upregulation of contractile ET{sub B} receptors by DSP was abrogated by U0126, SP600125, actinomycin D, and cycloheximide, suggesting that the underlying molecular mechanisms involved in this process include activation of MEK and JNK MAPK-mediated transcription and translation of new contractile ET{sub B} receptors. Thus, the MAPK-mediated upregulation of contractile ET{sub B

  9. Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton.

    PubMed

    Zhang, Xueying; Xu, Xiaoyang; Yu, Yujia; Chen, Chuan; Wang, Jing; Cai, Caiping; Guo, Wangzhen

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development, as well as their biotic and abiotic stress responses. As a nodal point of the MAPK cascade, the MKK gene family has not been systematically studied in cotton. Here, we identified 11 putative MKK genes in the Gossypium raimondii genome. Phylogenetic analysis showed that the MKKs were supported by architectures of conserved protein motifs. Expression patterns of MKKs under hormone treatments or abiotic stresses revealed their diverse functions in stress responses. Based on a yeast two hybrid, a total of 63 interactive pairs of MKKs and MAPKs were identified in cotton. Among these, 40 interactive pairs were newly identified compared to that reported previously in Arabidopsis. Integration analysis of the interaction network and expression patterns of MKK and MAPK family members revealed 13 potential MAPK signaling modules that are involved in the complicated cross-talk between hormones and abiotic stresses. Taken together, our data enhance the understanding of the evolution and function of MAPK cascades in cotton, and lay the foundation for the improvement of various defense responses that use MAPK signaling modules in the future. PMID:27417377

  10. Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton

    PubMed Central

    Zhang, Xueying; Xu, Xiaoyang; Yu, Yujia; Chen, Chuan; Wang, Jing; Cai, Caiping; Guo, Wangzhen

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development, as well as their biotic and abiotic stress responses. As a nodal point of the MAPK cascade, the MKK gene family has not been systematically studied in cotton. Here, we identified 11 putative MKK genes in the Gossypium raimondii genome. Phylogenetic analysis showed that the MKKs were supported by architectures of conserved protein motifs. Expression patterns of MKKs under hormone treatments or abiotic stresses revealed their diverse functions in stress responses. Based on a yeast two hybrid, a total of 63 interactive pairs of MKKs and MAPKs were identified in cotton. Among these, 40 interactive pairs were newly identified compared to that reported previously in Arabidopsis. Integration analysis of the interaction network and expression patterns of MKK and MAPK family members revealed 13 potential MAPK signaling modules that are involved in the complicated cross-talk between hormones and abiotic stresses. Taken together, our data enhance the understanding of the evolution and function of MAPK cascades in cotton, and lay the foundation for the improvement of various defense responses that use MAPK signaling modules in the future. PMID:27417377

  11. TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

    PubMed

    Gomes, Marco Túlio; Campos, Priscila Carneiro; Pereira, Guilherme de Sousa; Bartholomeu, Daniella Castanheira; Splitter, Gary; Oliveira, Sergio Costa

    2016-05-01

    Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9.

  12. Tiliroside, the major component of Agrimonia pilosa Ledeb ethanol extract, inhibits MAPK/JNK/p38-mediated inflammation in lipopolysaccharide-activated RAW 264.7 macrophages

    PubMed Central

    JIN, XIN; SONG, SHIQING; WANG, JING; ZHANG, QINGZHEN; QIU, FENG; ZHAO, FENG

    2016-01-01

    In the present study, the in vivo anti-inflammatory activity of Agrimonia pilosa Ledeb (AP) ethanol extract was confirmed in experimental animal models, including xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. Tiliroside, the major component of AP extract, was isolated and purified by high-performance liquid chromatography. The anti-inflammatory mechanism of tiliroside was then examined using lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells. An MTT assay was used to determine cytotoxicity and a Griess assay was used to determine nitric oxide (NO) production. Concentration levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme-linked immunosorbent assay. Protein expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), phosphorylated (p)-extracellular signal-regulated kinase (ERK) 1/2, p-c-Jun N-terminal kinases (JNK), p-p38 and inhibitor of κB-α were detected by western blot analysis. AP ethanol extract was revealed to inhibit xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. Tiliroside significantly suppressed the overproduction of NO (P<0.01), but revealed no notable inhibition of the release of TNF-α and IL-6. In addition, tiliroside significantly downregulated the elevated expression levels of iNOS and COX-2 induced by LPS (P<0.01). The phosphorylation of JNK and p38 proteins were also significantly inhibited (P<0.01), however, tiliroside exhibited no obvious inhibition on the phosphorylation of ERK 1/2 and the degradation of IκB-α protein. In conclusion, the anti-inflammatory molecular mechanism of tiliroside may involve the downregulation of iNOS and COX-2 protein expression levels, and the inactivation of mitogen-activated protein kinase (MAPK)/JNK, in addition to the MAPK/p38 signaling pathway. PMID:27347085

  13. A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

    PubMed Central

    Moreno, Ricardo D.

    2014-01-01

    Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis. PMID:25474107

  14. HBx-related long non-coding RNA DBH-AS1 promotes cell proliferation and survival by activating MAPK signaling in hepatocellular carcinoma.

    PubMed

    Huang, Jin-lan; Ren, Ting-yu; Cao, Shun-wang; Zheng, Shi-hao; Hu, Xiu-mei; Hu, Yan-wei; Lin, Li; Chen, Jing; Zheng, Lei; Wang, Qian

    2015-10-20

    Accumulating evidence supports an important role for the hepatitis B virus x protein (HBx) in the pathogenesis of hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC), but the underlying mechanisms are not entirely clear. Here, we identified a novel long noncoding RNA (lncRNA) DBH-AS1 involved in the HBx-mediated hepatocarcinogenesis. The levels of DBH-AS1 were positively correlated with hepatitis B surface antigen (HBsAg) and tumor size in HCC tissues. Functionally, transgenic expression of DBH-AS1 significantly enhanced cell proliferation and tumorigenesis, whereas short hairpin RNA knockdown of DBH-AS1 caused an inhibition of cell proliferation. Mechanistically, overexpression of DBH-AS1 induced cell cycle progression by accelerating G1/S and G2/M transition concomitantly with upregulation of CDK6, CCND1, CCNE1 and downregulation of p16, p21 and p27. We also found that enhanced DBH-AS1 expression inhibited serum starvation-induced apoptosis of HCC cells. In contrast, suppressed DBH-AS1 expression had opposite effects. Furthermore, DBH-AS1 was shown to activate MAPK pathway. We also provide evidence that DBH-AS1 could be significantly induced by HBx protein and markedly down-regulated by p53. Thus, we concluded that DBH-AS1 can be induced by HBx and inactivated by p53, and consequently promote cell proliferation and cell survival through activation of MAPK signaling in HCC. Our study suggests that DBH-AS1 acts as an oncogene for HCC. PMID:26393879

  15. A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts

    PubMed Central

    Sacristán-Reviriego, Almudena; Madrid, Marisa; Cansado, José; Martín, Humberto; Molina, María

    2014-01-01

    Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi. PMID:24465549

  16. Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

    SciTech Connect

    An, Caiyan; Sato, Koichi; Wu, Taoya; Bao, Muqiri; Bao, Liang; Tobo, Masayuki; Damirin, Alatangaole

    2015-05-01

    The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF. - Highlights: • Acidic pH and PDGF synergize to stimulate MEFs migration via Gi/p38MAPK pathway. • Extracellular acidification inhibits the

  17. Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

    PubMed

    Sampieri, Clara L; Nuttall, Robert K; Young, David A; Goldspink, Deborah; Clark, Ian M; Edwards, Dylan R

    2008-03-01

    The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.

  18. Time-dependent enhancement of inhibitory avoidance retention and MAPK activation by post-training infusion of nerve growth factor into CA1 region of hippocampus of adult rats.

    PubMed

    Walz, R; Lenz, G; Roesler, R; Vianna, M M; Martins, V; Brentani, R; Rodnight, R; Izquierdo, I

    2000-06-01

    Several studies have demonstrated that chronic intracerebroventricular nerve growth factor (NGF) infusion has a beneficial effect on cognitive performance of lesioned as well as old and developing animals. Here we investigate: (i) the effect of post-training infusion of NGF into the CA1 region of hippocampus on inhibitory avoidance (IA) retention in rats; (ii) the extension of the effect, in time and space, of NGF infusion into CA1 on the activity of mitogen-activated protein kinase (MAPK, syn: ERK1/2, p42/p44 MAPK). NGF was bilaterally injected into the CA1 regions of the dorsal hippocampus (0.05, 0.5 or 5.0 ng diluted in 0.5 microL of saline per side ) at 0, 120 or 360 min after IA training in rats. Retention testing was carried out 24 h after training. The injection of 5.0 and 0.5, but not 0.05, ng per side of NGF at 0 and 120 min after IA training enhanced IA retention. The highest dose used was ineffective when injected 360 min after training. The infusion of 0. 5 microL of NGF (5.0 ng) induced a significant enhancement of MAPK activity in hippocampal microslices; this enhancement was restricted to a volume with 0.8 mm radius at 30 min after injection. The MAPK activation was still seen 180 min after NGF infusion, although this value showed only a tendency. In conclusion, localized infusion of NGF into the CA1 region enhanced MAPK activity, restricted in time and space, and enhanced IA retention in a time- and dose-dependent manner.

  19. A novel lipopeptide from skin commensal activates TLR2/CD36-p38 MAPK signaling to increase antibacterial defense against bacterial infection.

    PubMed

    Li, Dongqing; Lei, Hu; Li, Zhiheng; Li, Hongquan; Wang, Yue; Lai, Yuping

    2013-01-01

    Staphylococcus epidermidis (S.epidermidis) plays important protective roles by directly producing or by stimulating hosts to produce antimicrobial peptides (AMPs) against pathogenic infections. Although several AMPs from S.epidermidis have been identified, molecules that stimulate hosts to produce AMPs remain largly unknown. Here we demonstrate that a new lipopeptide (named LP01) purified from S.epidermidis culture media has a unique structure with heneicosanoic acid (21 carbons) binding to lysine(11) of a peptide chain. In vitro LP01 increased the expression of β-defensin 2(hBD2) and hBD3 in neonatal human epidermal keratinocytes(NHEK), leading to increased capacity of cell lysates to inhibit the growth of S.aureus. In vivo LP01 induced the expression of mouse β-defensin 4(mBD4) to decrease the survival of local S.aureus in skin and systemic S.aureus survival in liver. The induction of beta-defensins by LP01 was dependent on TLR2 as Tlr2-deficient mice had decreased mBD4. Furthermore, knockdown of CD36 decreased the expression of hBD2 and hBD3, and p38 MAPK inhibitor significantly inhibited the expression of hBDs induced by LP01.Taken together, these findings demonstrate that lipopeptide LP01 from normal commensal S.epidermidis increases antimicrobial peptide hBD2 and hBD3 expression via the activation of TLR2/CD36-p38 MAPK, thus enhancing antimicrobial defense against pathogenic infections.

  20. Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways.

    PubMed

    Szegedi, Viktor; Juhász, Gábor; Zhang, Xiaoqun; Barkóczi, Balázs; Qi, Hongshi; Madeira, Alexandra; Kapus, Gábor; Svenningsson, Per; Spedding, Michael; Penke, Botond

    2011-12-01

    Impairments of cellular plasticity appear to underlie the pathophysiology of major depression. Recently, elevated levels of phosphorylated AMPA receptor were implicated in the antidepressant effect of various drugs. Here, we investigated the effects of an antidepressant, Tianeptine, on synaptic function and GluA1 phosphorylation using murine hippocampal slices and in vivo single-unit recordings. Tianeptine, but not imipramine, increased AMPA receptor-mediated neuronal responses both in vitro and in vivo, in a staurosporine-sensitive manner. Paired-pulse ratio was unaltered by Tianeptine, suggesting a postsynaptic site of action. Tianeptine, 10 μM, enhanced the GluA1-dependent initial phase of LTP, whereas 100 μM impaired the latter phases, indicating a critical role of GluA1 subunit phosphorylation in the excitation. Tianeptine rapidly increased the phosphorylation level of Ser(831)-GluA1 and Ser(845)-GluA1. Using H-89 and KN-93, we show that the activation of both PKA and CaMKII is critical in the effect of Tianeptine on AMPA responses. Moreover, the phosphorylation states of Ser(217/221)-MEK and Thr(183)/Tyr(185)-p42MAPK were increased by Tianeptine and specific kinase blockers of the MAPK pathways (PD 98095, SB 203580 and SP600125) prevented the effects of Tianeptine. Overall these data suggest that Tianeptine potentiates several signaling cascades associated with synaptic plasticity and provide further evidence that a major mechanism of action for Tianeptine is to act as an enhancer of glutamate neurotransmission via AMPA receptors.

  1. Triterpenoid Saponin W3 from Anemone flaccida Suppresses Osteoclast Differentiation through Inhibiting Activation of MAPKs and NF-κB Pathways.

    PubMed

    Kong, Xiangying; Yang, Yue; Wu, Wenbin; Wan, Hongye; Li, Xiaomin; Zhong, Michun; Su, Xiaohui; Jia, Shiwei; Lin, Na

    2015-01-01

    Excessive bone resorption by osteoclasts within inflamed joints is the most specific hallmark of rheumatoid arthritis. A. flaccida has long been used for the treatment of arthritis in folk medicine of China; however, the active ingredients responsible for the anti-arthritis effects of A. flaccida are still elusive. In this study, W3, a saponin isolated from the extract of A. flaccida was identified as the major active ingredient by using an osteoclast formation model induced by receptor activator of nuclear factor kappa-B ligand (RANKL). W3 dose-dependently suppressed the actin ring formation and lacunar resorption. Mechanistic investigation revealed that W3 inhibited the RANKL-induced TRAF6 expression, decreased phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and suppressed NF-κB p65 DNA binding activity. Furthermore, W3 almost abrogated the expression of c-Fos and nuclear factor of activated T cells (NFATc1). Therefore, our results suggest that W3 is a potential agent for treating lytic bone diseases although further evaluation in vivo and in clinical trials is needed. PMID:26327814

  2. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-κB and MAPK pathways.

    PubMed

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

    2011-11-18

    Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions. PMID:22037580

  3. Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon (Salmo salar) fed soy bean oil or fish oil based diets.

    PubMed

    Holen, E; Winterthun, S; Du, Z-Y; Krøvel, A V

    2011-01-01

    Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL-1β, TNF-α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL-1β, TNF-α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish's ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently.

  4. Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumor-initiation triggering mitochondrial death pathway.

    PubMed

    Bhui, Kulpreet; Prasad, Sahdeo; George, Jasmine; Shukla, Yogeshwer

    2009-09-18

    Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple (Ananas cosmosus), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of approximately 65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-kappaB) was recorded, as phosphorylation and consequent degradation of I kappa B alpha was blocked by bromelain. Also, bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by bromelain is a coherent approach in achieving chemoprevention.

  5. Anti-Inflammatory Effect of Mangostenone F in Lipopolysaccharide-Stimulated RAW264.7 Macrophages by Suppressing NF-κB and MAPK Activation

    PubMed Central

    Cho, Byoung Ok; Ryu, Hyung Won; So, Yangkang; Lee, Chang Wook; Jin, Chang Hyun; Yook, Hong Sun; Jeong, Yong Wook; Park, Jong Chun; Jeong, Il Yun

    2014-01-01

    Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-κB luciferase activity and NF-κB DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-κB activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages. PMID:25143806

  6. Interleukin-10 Enhances the Intestinal Epithelial Barrier in the Presence of Corticosteroids through p38 MAPK Activity in Caco-2 Monolayers: A Possible Mechanism for Steroid Responsiveness in Ulcerative Colitis

    PubMed Central

    Lorén, Violeta; Cabré, Eduard; Ojanguren, Isabel; Domènech, Eugeni; Pedrosa, Elisabet; García-Jaraquemada, Arce; Mañosa, Miriam; Manyé, Josep

    2015-01-01

    Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn’s disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis. PMID:26090671

  7. Suomi NPP VIIRS active fire product status

    NASA Astrophysics Data System (ADS)

    Ellicott, E. A.; Csiszar, I. A.; Schroeder, W.; Giglio, L.; Wind, B.; Justice, C. O.

    2012-12-01

    We provide an overview of the evaluation and development of the Active Fires product derived from the Visible Infrared Imager Radiometer Suite (VIIRS) sensor on the Suomi National Polar-orbiting Partnership (SNPP) satellite during the first year of on-orbit data. Results from the initial evaluation of the standard SNPP Active Fires product, generated by the SNPP Interface Data Processing System (IDPS), supported the stabilization of the VIIRS Sensor Data Record (SDR) product. This activity focused in particular on the processing of the dual-gain 4 micron VIIRS M13 radiometric measurements into 750m aggregated data, which are fundamental for active fire detection. Following the VIIRS SDR product's Beta maturity status in April 2012, correlative analysis between VIIRS and near-simultaneous fire detections from the Moderate Resolution Imaging Spectroradiometer (MODIS) on the NASA Earth Observing System Aqua satellite confirmed the expected relative detection rates driven primarily by sensor differences. The VIIRS Active Fires Product Development and Validation Team also developed a science code that is based on the latest MODIS Collection 6 algorithm and provides a full spatially explicit fire mask to replace the sparse array output of fire locations from a MODIS Collection 4 equivalent algorithm in the current IDPS product. The Algorithm Development Library (ADL) was used to support the planning for the transition of the science code into IDPS operations in the future. Product evaluation and user outreach was facilitated by a product website that provided end user access to fire data in user-friendly format over North America as well as examples of VIIRS-MODIS comparisons. The VIIRS fire team also developed an experimental product based on 375m VIIRS Imagery band measurements and provided high quality imagery of major fire events in US. By August 2012 the IDPS product achieved Beta maturity, with some known and documented shortfalls related to the processing of

  8. Distal retinal ganglion cell axon transport loss and activation of p38 MAPK stress pathway following VEGF-A antagonism

    PubMed Central

    Foxton, R; Osborne, A; Martin, K R; Ng, Y-S; Shima, D T

    2016-01-01

    There is increasing evidence that VEGF-A antagonists may be detrimental to neuronal health following ocular administration. Here we investigated firstly the effects of VEGF-A neutralization on retinal neuronal survival in the Ins2Akita diabetic and JR5558 spontaneous choroidal neovascularization (CNV) mice, and then looked at potential mechanisms contributing to cell death. We detected elevated apoptosis in the ganglion cell layer in both these models following VEGF-A antagonism, indicating that even when vascular pathologies respond to treatment, neurons are still vulnerable to reduced VEGF-A levels. We observed that retinal ganglion cells (RGCs) seemed to be the cells most susceptible to VEGF-A antagonism, so we looked at anterograde transport in these cells, due to their long axons requiring optimal protein and organelle trafficking. Using cholera toxin B-subunit tracer studies, we found a distal reduction in transport in the superior colliculus following VEGF-A neutralization, which occurred prior to net RGC loss. This phenomenon of distal transport loss has been described as a feature of early pathological changes in glaucoma, Alzheimer's and Parkinson's disease models. Furthermore, we observed increased phosphorylation of p38 MAPK and downstream Hsp27 stress pathway signaling in the retinas from these experiments, potentially providing a mechanistic explanation for our findings. These experiments further highlight the possible risks of using VEGF-A antagonists to treat ocular neovascular disease, and suggest that VEGF-A may contribute to the maintenance and function of axonal transport in neurons of the retina. PMID:27148685

  9. Distal retinal ganglion cell axon transport loss and activation of p38 MAPK stress pathway following VEGF-A antagonism.

    PubMed

    Foxton, R; Osborne, A; Martin, K R; Ng, Y-S; Shima, D T

    2016-01-01

    There is increasing evidence that VEGF-A antagonists may be detrimental to neuronal health following ocular administration. Here we investigated firstly the effects of VEGF-A neutralization on retinal neuronal survival in the Ins2(Akita) diabetic and JR5558 spontaneous choroidal neovascularization (CNV) mice, and then looked at potential mechanisms contributing to cell death. We detected elevated apoptosis in the ganglion cell layer in both these models following VEGF-A antagonism, indicating that even when vascular pathologies respond to treatment, neurons are still vulnerable to reduced VEGF-A levels. We observed that retinal ganglion cells (RGCs) seemed to be the cells most susceptible to VEGF-A antagonism, so we looked at anterograde transport in these cells, due to their long axons requiring optimal protein and organelle trafficking. Using cholera toxin B-subunit tracer studies, we found a distal reduction in transport in the superior colliculus following VEGF-A neutralization, which occurred prior to net RGC loss. This phenomenon of distal transport loss has been described as a feature of early pathological changes in glaucoma, Alzheimer's and Parkinson's disease models. Furthermore, we observed increased phosphorylation of p38 MAPK and downstream Hsp27 stress pathway signaling in the retinas from these experiments, potentially providing a mechanistic explanation for our findings. These experiments further highlight the possible risks of using VEGF-A antagonists to treat ocular neovascular disease, and suggest that VEGF-A may contribute to the maintenance and function of axonal transport in neurons of the retina. PMID:27148685

  10. Involvement of MAPK ERK activation in upregulation of water channel protein aquaporin 1 in a mouse model of Bell's palsy.

    PubMed

    Fang, Fan; Liu, Cai-Yue; Zhang, Jie; Zhu, Lie; Qian, Yu-Xin; Yi, Jing; Xiang, Zheng-Hua; Wang, Hui; Jiang, Hua

    2015-05-01

    The aim of this study is to immunolocalize the aquaporin 1 water channel protein (AQP1) in Schwann cells of idiopathic facial nerve and explore its possible role during the development of facial palsy induced by herpes simplex virus type 1 (HSV-1). HSV-1 was inoculated into the surface of posterior auricle of mouse to establish a paralyzed animal model. In HSV-1-induced facial palsy mice, protein levels of AQP1 significantly increased on the 9th to 16th day after inoculation of HSV-1. The upregulation of AQP1 was closely related to the intratemporal facial nerve edema in facial nerve canal, which was also consistent with the symptom of facial palsy in mice. In a hypoxia model of Schwann cells in vitro, we found that U0126, an ERK antagonist, inhibited not only morphological changes of cultures Schwann cells but also upregulation of both AQP1 and phosphorylated ERK. Combined with increased phosphorylated ERK in HSV-1-induced facial palsy mice, we inferred that ERK MAPK pathway might also be involved in increased AQP1 in mouse model of Bell's palsy. Although the precise mechanism needs to be further explored, our findings suggest that AQP1 in Schwann cells of intratemporal facial nerve is involved in the evolution of facial palsy induced by HSV-1 and may play an important role in the pathogenesis of this disease. AQP1 might be a potential target, and the ERK antagonist U0126 could be a new drug for the treatment of HSV-1-induced Bell's palsy in an early stage. PMID:25527444

  11. MAPK genes interact with diet and lifestyle factors to alter risk of breast cancer: the Breast Cancer Health Disparities Study.

    PubMed

    Slattery, Martha L; Lundgreen, Abbie; John, Esther M; Torres-Mejia, Gabriela; Hines, Lisa; Giuliano, Anna R; Baumgartner, Kathy B; Stern, Mariana C; Wolff, Roger K

    2015-01-01

    Mitogen-activated protein kinases (MAPK) are integration points for multiple biochemical signals. We evaluated 13 MAPK genes with breast cancer risk and determined if diet and lifestyle factors mediated risk. Data from 3 population-based case-control studies conducted in Southwestern United States, California, and Mexico included 4183 controls and 3592 cases. Percent Indigenous American (IA) ancestry was determined from 104 ancestry informative markers. The adaptive rank truncated product (ARTP) was used to determine the significance of each gene and the pathway with breast cancer risk, by menopausal status, genetic ancestry level, and estrogen receptor (ER)/progesterone receptor (PR) strata. MAP3K9 was associated with breast cancer overall (P(ARTP) = 0.02) with strongest association among women with the highest IA ancestry (P(ARTP) = 0.04). Several SNPs in MAP3K9 were associated with ER+/PR+ tumors and interacted with dietary oxidative balance score (DOBS), dietary folate, body mass index (BMI), alcohol consumption, cigarette smoking, and a history of diabetes. DUSP4 and MAPK8 interacted with calories to alter breast cancer risk; MAPK1 interacted with DOBS, dietary fiber, folate, and BMI; MAP3K2 interacted with dietary fat; and MAPK14 interacted with dietary folate and BMI. The patterns of association across diet and lifestyle factors with similar biological properties for the same SNPs within genes provide support for associations.

  12. Catalase, but not MnSOD, inhibits glucose deprivation-activated ASK1-MEK-MAPK signal transduction pathway and prevents relocalization of Daxx: hydrogen peroxide as a major second messenger of metabolic oxidative stress.

    PubMed

    Song, Jae J; Lee, Yong J

    2003-10-01

    Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation-induced cytotoxicity and c-Jun N-terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU-145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H(2)O(2)) rather than superoxide anion (O(2) (*-)) acts as a second messenger of metabolic oxidative stress to activate the ASK1-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-mitogen-activated protein kinase (MAPK) signal transduction pathway.

  13. Lentinan mitigates therarubicin-induced myelosuppression by activating bone marrow-derived macrophages in an MAPK/NF-κB-dependent manner.

    PubMed

    Liu, Qiang; Dong, Lei; Li, Hong; Yuan, Jia; Peng, Yuping; Dai, Shejiao

    2016-07-01

    Bone marrow (BM) suppression (also known as myelosuppression) is the most common and most severe side-effect of therarubicin (THP) and thereby limits the clinical application of this anticancer agent. Lentinan (LNT), a glucan extracted from dried shiitake mushrooms (Lentinula edodes), exhibits a variety of pharmacological activities. The objectives of the present study were to determine the effect of LNT on the myelosuppression of THP-treated mice and to examine the pharmacological mechanism of these effects. In vivo experiments indicated that non-cytotoxic levels of LNT strongly increased blood myeloperoxidase (MPO) activity; improved BM structural injuries; increased the numbers of leukocytes and neutrophils in the blood and BM; elevated the blood levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF); and reduced the self-healing period in THP-treated mice. In vitro experiments indicated that LNT increased the viability of BM-derived macrophages (BMDMs) in a time- and dose-dependent manner without toxic side-effects and markedly increased the release of G-CSF, GM-CSF and M-CSF by BMDMs. Further analyses revealed that LNT activated the NF-κB and MAPK signalling pathways and promoted the nuclear import of p65 and that BAY 11-7082 (a specific inhibitor of NF-κB) suppressed the release of G-CSF, GM-CSF and M-CSF. Furthermore, we found that U0126, SB203580 and SP600125 (specific inhibitors of ERK, p38 and JNK, respectively) markedly inhibited the IKK/IκB/NF-κB-dependent release of G-CSF, GM-CSF and M-CSF. In conclusion, LNT induces the production of G-CSF, GM-CSF and M-CSF by activating the MAPK/NF-κB signalling pathway in BM cells, thereby mitigating THP-induced myelosuppression. PMID:27121155

  14. Roles of MAPK pathway activation during cytokine induction in BEAS-2B cells exposed to fine World Trade Center (WTC) dust.

    PubMed

    Wang, Shang; Prophete, Colette; Soukup, Joleen M; Chen, Lung-Chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Cohen, Mitchell D; Chen, Haobin

    2010-01-01

    The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter (PM) into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM₂.₅ fraction or WTC₂.₅). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the mitogen-activated protein kinase (MAPK) signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC₂.₅. Our results showed that exposure to WTC₂.₅ for 5 hr increased interleukin-6 (IL-6) mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, cytokine multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal protein kinase, signaling pathways were found to be activated in cells exposed to WTC₂.₅. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC₂.₅; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC₂.₅-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other

  15. Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

    PubMed Central

    Abou-Samra, Abdul B.

    2012-01-01

    Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0–40 min) of LLCP-K1 cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression. PMID:22414806

  16. Roles of MAPK Pathway Activation During Cytokine Induction in BEAS-2B Cells Exposed to Fine World Trade Center (WTC) Dust

    PubMed Central

    Wang, Shang; Prophete, Colette; Soukup, Joleen M.; Chen, Lung-chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Chen, Haobin

    2014-01-01

    The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM2.5 fraction or WTC2.5). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the MAPK signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC2.5. Our results showed that exposure to WTC2.5 for 5 hr increased IL-6 mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, Cytokine Multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both ERK and p38, but not JNK, signaling pathways were found to be activated in cells exposed to WTC2.5. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC2.5; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC2.5-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other inflammation-associated respiratory dysfunctions in the individuals exposed to the dusts released from the WTC collapse. PMID:20731619

  17. Notoginsenoside Rb1 inhibits activation of ERK and p38 MAPK pathways induced by hypoxia and hypercapnia

    PubMed Central

    QIU, XIAOXIAO; ZHENG, MENGXIAO; SONG, DONG; HUANG, LINJING; TANG, LANLAN; YING, LEI; WANG, WANTIE

    2016-01-01

    The aim of the present study was to investigate the effect of notoginsenoside Rb1 (Rb1) on the ERK and p38 MAPK pathways in primary cultured pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia and hypercapnia, in order to elucidate the mechanism underlying the effect of Rb1 on hypoxia and hypercapnia-induced pulmonary vasoconstriction (HHPV). PASMCs were isolated from Sprague-Dawley rats. The cells were divided into five groups: Normal (N), hypoxia and hypercapnia (H), RbL, RbM and RbH groups. N group cells were cultured under 5% CO2 and 21% O2. H, RbL, RbM and RbH groups were cultured under 6% CO2 and 1% O2. Prior to the hypoxia and hypercapnia exposure, RbL, RbM and RbH groups were treated with 8, 40 and 100 mg/ml Rb1 for 30 min, respectively. Phosphorylated extracellular signal-regulated kinase (P-ERK) and P-p38 protein, and ERK1/2 and p38 mRNA expression levels were detected using western blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, respectively. The correlations between P-ERK protein and ERK1/2 mRNA, and between P-p38 protein and p38 mRNA were evaluated. Results of western blot and RT-PCR showed hypoxia and hypercapnia increased P-ERK and P-p38 protein, and ERK1/2 mRNA, respectively (P<0.05). Rb1 suppressed the increased P-ERK and P-p38 protein, and ERK1/2 and p38 mRNA by hypoxia and hypercapnia (P<0.05). P-ERK protein was positively correlated with ERK1 (r=0.5, P<0.01) and ERK2 mRNA (r=0.977, P<0.01). P-p38 protein was positively correlated with p38 mRNA (r=0.884, P<0.01). Thus, the present results indicate that Rb1 may ameliorate HHPV by suppressing ERK and p38 pathways. The study provides an experimental basis for investigating the clinical use of Rb1 in the management of HHPV-related disorders. PMID:27313674

  18. Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

    PubMed

    Tsai, Chung-Che; Kuo, Ting-Yu; Hong, Zhi-Wei; Yeh, Ying-Chieh; Shih, Kuo-Shun; Du, Shin-Yi; Fu, Hua-Wen

    2015-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.

  19. Novel human neutrophil agonistic properties of arsenic trioxide: involvement of p38 mitogen-activated protein kinase and/or c-jun NH2-terminal MAPK but not extracellular signal-regulated kinases-1/2.

    PubMed

    Binet, François; Girard, Denis

    2008-12-01

    Arsenic trioxide (ATO) is known for treating acute promyelocytic leukemia and for inducing apoptosis and mitogen-activated protein kinases (MAPKs) in promyelocytes and cancer cells. We recently reported that ATO induces neutrophil apoptosis. The aim of this study was to establish whether or not ATO recruits MAPKs in neutrophils, as well as to further investigate its agonistic properties. We found that ATO activates p38 and that, unlike H2O2, this response was not inhibited by exogenous catalase. Also, we demonstrated that ATO-induced p38 activation occurs before H2O2 generation and without a calcium burst. We next established that ATO recruits c-jun NH2-terminal (JNK) but not extracellular signal-regulated kinase 1 and 2 (Erk-1/2). Using pharmacological inhibitors, we found that the proapoptotic activity of ATO occurs by a MAPK-independent mechanism. In contrast, the ability of ATO to enhance adhesion, migration, phagocytosis, release, and activity of gelatinase and degranulation of secretory, specific, and gelatinase, but not azurophilic granules, is dependent upon activation of p38 and/or JNK. This is the first study establishing that ATO possesses important agonistic properties in human neutrophils. Given the central role of neutrophils in various inflammatory disorders, we propose that ATO might have broader therapeutic implications in clinics, especially for regulating inflammation.

  20. The Neuroprotection of Liraglutide Against Ischaemia-induced Apoptosis through the Activation of the PI3K/AKT and MAPK Pathways

    PubMed Central

    Zhu, Huili; Zhang, Yusheng; Shi, Zhongshan; Lu, Dan; Li, Tingting; Ding, Yan; Ruan, Yiwen; Xu, Anding

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases glucose-dependent insulin secretion to reduce the glucose level. Liraglutide, a long-acting GLP-1 analogue, has been found to have neuroprotective action in various experimental models. However, the protective mechanisms of liraglutide in ischaemic stroke remain unclear. Here, we demonstrated that liraglutide significantly decreased the infarct volume, improved neurologic deficits, and lowered stress-related hyperglycaemia without causing hypoglycaemia in a rat model of middle cerebral artery occlusion (MCAO). Liraglutide inhibited cell apoptosis by reducing excessive reactive oxygen species (ROS) and improving the function of mitochondria in neurons under oxygen glucose deprivation (OGD) in vitro and MCAO in vivo. Liraglutide up-regulated the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinases (ERK) and inhibited the phosphorylation of c-jun-NH2-terminal kinase (JNK) and p38. Moreover, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and/or the ERK inhibitor U0126 counteracted the protective effect of liraglutide. Taken together, these results suggest that liraglutide exerts neuroprotective action against ischaemia-induced apoptosis through the reduction of ROS and the activation of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways. Therefore, liraglutide has therapeutic potential for patients with ischaemic stroke, especially those with Type 2 diabetes mellitus or stress hyperglycaemia. PMID:27240461

  1. Ozone (O3) elicits neurotoxicity in spinal cord neurons (SCNs) by inducing ER Ca(2+) release and activating the CaMKII/MAPK signaling pathway.

    PubMed

    Li, Yun; Lin, Xiaowen; Zhao, XueJun; Xie, Juntian; JunNan, Wang; Sun, Tao; Fu, Zhijian

    2014-11-01

    Ozone (O3) is widely used in the treatment of spinal cord related diseases. Excess or accumulation of this photochemical air can however be neurotoxic. In this study, in vitro cultured Wister rat spinal cord neurons (SCNs) were used to investigate the detrimental effects and underlying mechanisms of O3. Ozone in a dose-dependent manner inhibited cell viability at a range of 20 to 500 μg/ml, with the dose at 40 μg/ml resulting in a decrease of cell viability to 75%. The cell death after O3 exposure was related to endoplasmic reticulum (ER) calcium (Ca(2+)) release. Intracellular Ca(2+) chelator, ER stabilizer (inositol 1,4,5-trisphosphate receptor (IP3R) antagonist and ryanodine receptor (RyR) antagonist) and calcium/calmodulin-dependent protein kinase II (CaMKII) antagonist could effectively block Ca(2+) mobilization and inhibit cell death following 40 μg/ml O3 exposure. In addition, ER Ca(2+) release due to O3 exposure enhanced phospho-p38 and phospho-JNK levels and apoptosis of SCNs through activating CaMKII. Based on these results, we confirm that ozone elicits neurotoxicity in SCNs via inducing ER Ca(2+) release and activating CaMKII/MAPK signaling pathway. Therefore, physicians should get attention to the selection of treatment concentrations of oxygen/ozone. And, approaches, such as chelating intracellular Ca(2+) and stabilizing neuronal Ca(2+) homeostasis could effectively ameliorate the neurotoxicity of O3.

  2. Wogonin suppresses TNF-{alpha}-induced MMP-9 expression by blocking the NF-{kappa}B activation via MAPK signaling pathways in human aortic smooth muscle cells

    SciTech Connect

    Lee, Syng-Ook; Jeong, Yun-Jeong; Yu, Mi Hee; Lee, Ji-Won; Hwangbo, Mi Hyang; Kim, Cheorl-Ho; Lee, In-Seon . E-mail: inseon@kmu.ac.kr

    2006-12-08

    Matrix metalloproteinase-9 (MMP-9) plays a major role in the pathogenesis of atherosclerosis and restenosis by regulating both migration and proliferation of vascular smooth muscle cells (VSMC) after an arterial injury. In this study, we examined the inhibitory effect of three major flavonoids in Scutellariae Radix, baicalin, baicalein, and wogonin, on TNF-{alpha}-induced MMP-9 expression in human aortic smooth muscle cells (HASMC). Wogonin, but not baicalin and baicalein, significantly and selectively suppressed TNF-{alpha}-induced MMP-9 expression in HASMC. Reporter gene, electrophoretic mobility shift, and Western blotting assays showed that wogonin inhibits MMP-9 gene transcriptional activity by blocking the activation of NF-{kappa}B via MAPK signaling pathways. Moreover, the Matrigel migration assay showed that wogonin reduced TNF-{alpha}-induced HASMC migration. These results suggest that wogonin effectively suppresses TNF-{alpha}-induced HASMC migration through the selective inhibition of MMP-9 expression and represents a potential agent for the prevention of vascular disorders related to the migration of VSMC.

  3. Atf1-Pcr1-M26 Complex Links Stress-activated MAPK and cAMP-dependent Protein Kinase Pathways via Chromatin Remodeling of cgs2+*

    PubMed Central

    Davidson, Mari K.; Shandilya, Harish K.; Hirota, Kouji; Ohta, Kunihiro; Wahls, Wayne P.

    2011-01-01

    Although co-ordinate interaction between different signal transduction pathways is essential for developmental decisions, interpathway connections are often obscured and difficult to identify due to cross-talk. Here signals from the fission yeast stress-activated MAPK Spc1 are shown to regulate Cgs2, a negative regulator of the cAMP-dependent protein kinase (protein kinase A) pathway. Pathway integration is achieved via Spc1-dependent binding of Atf1-Pcr1 heterodimer to an M26 DNA site in the cgs2+ promoter, which remodels chromatin to regulate expression of cgs2+ and targets downstream of protein kinase A. This direct interpathway connection co-ordinates signals of nitrogen and carbon source depletion to affect a G0 cell-cycle checkpoint and sexual differentiation. The Atf1-Pcr1-M26 complex-dependent chromatin remodeling provides a molecular mechanism whereby Atf1-Pcr1 heterodimer can function differentially as either a transcriptional activator, or as a transcriptional repressor, or as an inducer of meiotic recombination. We also show that the Atf1-Pcr1-M26 complex functions as both an inducer and repressor of chromatin remodeling, which provides a way for various chromatin remodeling-dependent effector functions to be regulated. PMID:15448137

  4. Atf1-Pcr1-M26 complex links stress-activated MAPK and cAMP-dependent protein kinase pathways via chromatin remodeling of cgs2+.

    PubMed

    Davidson, Mari K; Shandilya, Harish K; Hirota, Kouji; Ohta, Kunihiro; Wahls, Wayne P

    2004-12-01

    Although co-ordinate interaction between different signal transduction pathways is essential for developmental decisions, interpathway connections are often obscured and difficult to identify due to cross-talk. Here signals from the fission yeast stress-activated MAPK Spc1 are shown to regulate Cgs2, a negative regulator of the cAMP-dependent protein kinase (protein kinase A) pathway. Pathway integration is achieved via Spc1-dependent binding of Atf1-Pcr1 heterodimer to an M26 DNA site in the cgs2+ promoter, which remodels chromatin to regulate expression of cgs2+ and targets downstream of protein kinase A. This direct interpathway connection co-ordinates signals of nitrogen and carbon source depletion to affect a G0 cell-cycle checkpoint and sexual differentiation. The Atf1-Pcr1-M26 complex-dependent chromatin remodeling provides a molecular mechanism whereby Atf1-Pcr1 heterodimer can function differentially as either a transcriptional activator, or as a transcriptional repressor, or as an inducer of meiotic recombination. We also show that the Atf1-Pcr1-M26 complex functions as both an inducer and repressor of chromatin remodeling, which provides a way for various chromatin remodeling-dependent effector functions to be regulated. PMID:15448137

  5. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts.

    PubMed

    Lu, Jing; Guo, Jia-Hui; Tu, Xue-Liang; Zhang, Chao; Zhao, Mei; Zhang, Quan-Wu; Gao, Feng-Hou

    2016-01-01

    Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced. PMID:27486852

  6. Sea cucumber peptides exert anti-inflammatory activity through suppressing NF-κB and MAPK and inducing HO-1 in RAW264.7 macrophages.

    PubMed

    Song, Jiajia; Li, Tiange; Cheng, Xue; Ji, Xiaomin; Gao, Dongxiao; Du, Min; Jiang, Naiyi; Liu, Xueling; Mao, Xueying

    2016-06-15

    The anti-inflammatory effect of sea cucumber peptides (SCP) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages was tested. SCP significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression without affecting the cell viability. The mRNA expression of LPS-induced inflammatory cytokines including tumour necrosis factor-α, interleukin (IL)-1β and IL-6 was suppressed. SCP inhibited LPS-induced degradation of the inhibitor of κBα (IκBα) and nuclear transposition of NF-κB p65, resulting in decreased NF-κB transactivation. Moreover, SCP suppressed the LPS-induced phosphorylation of JNK, ERK and p38. In addition, the expression of heme oxygenase (HO)-1 in macrophages was up-regulated by SCP in a dose-dependent manner. The inhibition effect of SCP on the mRNA expression of LPS-induced inflammatory cytokines was partially reversed by co-treatment with a HO-1 inhibitor. The SCP with anti-inflammatory activity was made up of low-molecular-weight peptides rich in glycine, glutamic acid and aspartic acid. Collectively, these results demonstrate that SCP exerts anti-inflammatory function through inhibiting NF-κB and MAPK activation and inducing HO-1 expression in macrophages.

  7. Sea cucumber peptides exert anti-inflammatory activity through suppressing NF-κB and MAPK and inducing HO-1 in RAW264.7 macrophages.

    PubMed

    Song, Jiajia; Li, Tiange; Cheng, Xue; Ji, Xiaomin; Gao, Dongxiao; Du, Min; Jiang, Naiyi; Liu, Xueling; Mao, Xueying

    2016-06-15

    The anti-inflammatory effect of sea cucumber peptides (SCP) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages was tested. SCP significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression without affecting the cell viability. The mRNA expression of LPS-induced inflammatory cytokines including tumour necrosis factor-α, interleukin (IL)-1β and IL-6 was suppressed. SCP inhibited LPS-induced degradation of the inhibitor of κBα (IκBα) and nuclear transposition of NF-κB p65, resulting in decreased NF-κB transactivation. Moreover, SCP suppressed the LPS-induced phosphorylation of JNK, ERK and p38. In addition, the expression of heme oxygenase (HO)-1 in macrophages was up-regulated by SCP in a dose-dependent manner. The inhibition effect of SCP on the mRNA expression of LPS-induced inflammatory cytokines was partially reversed by co-treatment with a HO-1 inhibitor. The SCP with anti-inflammatory activity was made up of low-molecular-weight peptides rich in glycine, glutamic acid and aspartic acid. Collectively, these results demonstrate that SCP exerts anti-inflammatory function through inhibiting NF-κB and MAPK activation and inducing HO-1 expression in macrophages. PMID:27220344

  8. Non-mutagenic Suppression of Enterocyte Ferroportin 1 by Chemical Ribosomal Inactivation via p38 Mitogen-activated Protein Kinase (MAPK)-mediated Regulation: EVIDENCE FOR ENVIRONMENTAL HEMOCHROMATOSIS.

    PubMed

    Oh, Chang-Kyu; Park, Seong-Hwan; Kim, Juil; Moon, Yuseok

    2016-09-16

    Iron transfer across the basolateral membrane of an enterocyte into the circulation is the rate-limiting step in iron absorption and is regulated by various pathophysiological factors. Ferroportin (FPN), the only known mammalian iron exporter, transports iron from the basolateral surface of enterocytes, macrophages, and hepatocytes into the blood. Patients with genetic mutations in FPN or repeated blood transfusion develop hemochromatosis. In this study, non-mutagenic ribosomal inactivation was assessed as an etiological factor of FPN-associated hemochromatosis in enterocytes. Non-mutagenic chemical ribosomal inactivation disrupted iron homeostasis by regulating expression of the iron exporter FPN-1, leading to intracellular accumulation in enterocytes. Mechanistically, a xenobiotic insult stimulated the intracellular sentinel p38 MAPK signaling pathway, which was positively involved in FPN-1 suppression by ribosomal dysfunction. Moreover, ribosomal inactivation-induced iron accumulation in Caenorhabditis elegans as a simplified in vivo model for gut nutrition uptake was dependent on SEK-1, a p38 kinase activator, leading to suppression of FPN-1.1 expression and iron accumulation. In terms of gene regulation, ribosomal stress-activated p38 signaling down-regulated NRF2 and NF-κB, both of which were positive transcriptional regulators of FPN-1 transcription. This study provides molecular evidence for the modulation of iron bioavailability by ribosomal dysfunction as a potent etiological factor of non-mutagenic environmental hemochromatosis in the gut-to-blood axis.

  9. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts

    PubMed Central

    Zhang, Chao; Zhao, Mei; Zhang, Quan-Wu; Gao, Feng-Hou

    2016-01-01

    Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced. PMID:27486852

  10. Atf1-Pcr1-M26 complex links stress-activated MAPK and cAMP-dependent protein kinase pathways via chromatin remodeling of cgs2+.

    PubMed

    Davidson, Mari K; Shandilya, Harish K; Hirota, Kouji; Ohta, Kunihiro; Wahls, Wayne P

    2004-12-01

    Although co-ordinate interaction between different signal transduction pathways is essential for developmental decisions, interpathway connections are often obscured and difficult to identify due to cross-talk. Here signals from the fission yeast stress-activated MAPK Spc1 are shown to regulate Cgs2, a negative regulator of the cAMP-dependent protein kinase (protein kinase A) pathway. Pathway integration is achieved via Spc1-dependent binding of Atf1-Pcr1 heterodimer to an M26 DNA site in the cgs2+ promoter, which remodels chromatin to regulate expression of cgs2+ and targets downstream of protein kinase A. This direct interpathway connection co-ordinates signals of nitrogen and carbon source depletion to affect a G0 cell-cycle checkpoint and sexual differentiation. The Atf1-Pcr1-M26 complex-dependent chromatin remodeling provides a molecular mechanism whereby Atf1-Pcr1 heterodimer can function differentially as either a transcriptional activator, or as a transcriptional repressor, or as an inducer of meiotic recombination. We also show that the Atf1-Pcr1-M26 complex functions as both an inducer and repressor of chromatin remodeling, which provides a way for various chromatin remodeling-dependent effector functions to be regulated.

  11. Activation of microglia by endotoxin suppresses the secretion of glial cell line-derived neurotrophic factor (GDNF) through the action of protein kinase C alpha (PKCalpha) and mitogen-activated protein kinases (MAPKS).

    PubMed

    Matsushita, Yuichi; Nakajima, Kazuyuki; Tohyama, Yoko; Kurihara, Tadashi; Kohsaka, Shinichi

    2008-07-01

    The ability of microglia to produce/secrete glial cell line-derived neurotrophic factor (GDNF) in vitro was examined. Immunoblotting analysis revealed that nonstimulated microglia release limited amounts of GDNF with molecular sizes of 14 and 17 kDa. However, the secreted amounts significantly decreased when the microglia were activated with the endotoxin lipopolysaccharide (LPS). Comparison of the amounts of GDNF in the cells and the conditioned medium between the nonstimulated microglia and LPS-stimulated microglia clarified that the secretion of GDNF, but not its production, is strongly suppressed when the microglia are activated with LPS. The inhibitor experiments suggested that the GDNF secretion is depressed by a signaling cascade associated with protein kinase C alpha (PKCalpha) and/or mitogen-activated protein kinases (MAPKs). As expected from the above results, a PKC activator suppressed the secretion of GDNF in nonstimulated microglia. Taken together, these results demonstrated that microglia have the ability to produce and secrete GDNF in vitro, and that the secretion is suppressed by stimulation with endotoxin, probably due to a signaling mechanism involving PKCalpha and/or MAPKs.

  12. far4, far5, and far6 define three genes required for efficient activation of MAPKs Fus3 and Kss1 and accumulation of glycogen.

    PubMed

    Cherkasova, V; Elion, E A

    2001-08-01

    In Saccharomyces cerevisiae, mating pheromones induce G1 arrest through the activation of two MAP kinases, Fus3 and Kss1. Here we report the isolation of three mutants, far4, far5, and far6, that have the novel phenotype of regulating both the activity of Fus3 and Kss1 and the accumulation of glycogen. A far4 mutation constitutively activates Fus3 and Kss1, reduces glycogen, and blocks G1 arrest in the presence of alpha factor. In contrast, far5 and far6 mutations increase glycogen and reduce activation of Fus3 and Kss1 by pheromone. far4, far5, and far6 are recessive and not allelic to FAR1, FAR3, or 14 genes known to regulate the pheromone response. Non-allelic noncomplementation occurs between far6 and both far4 and far5, suggesting that FAR6 functionally interacts with FAR4 and FAR5. Additional observations suggest that FAR4 has functional overlap with FAR3, which we also find to regulate glycogen accumulation. Our results suggest that the activation of the mating MAPK cascade and subsequent G1 arrest is influenced by a signal transduction pathway that regulates glycogen. In support of this possibility, we find that Fus3 is activated to a greater extent in a "wimp" strain with defective protein kinase A. Finally, BIM1 and BIK1 have been identified as CEN suppressors of far5, suggesting that the microtubule apparatus may regulate the ability of the pheromone response pathway to promote G1 arrest. PMID:11570512

  13. [RELATIONSHIP BETWEEN p53/p21/Rb AND MAPK SIGNALING PATHWAYS IN HUMAN ENDOMETRIUM-DERIVED STEM CELLS UNDER OXIDATIVE STRESS].

    PubMed

    Deryabin, P I; Borodkina, A V; Nikolsky, N N; Burova, E B

    2015-01-01

    Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors--pifithrin-α (PFT), U0126, SB203580 and SP600125 to "switch off" one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway.

  14. p38 MAPK Signaling in Pemphigus: Implications for Skin Autoimmunity

    PubMed Central

    Mavropoulos, Athanasios; Orfanidou, Timoklia; Liaskos, Christos; Smyk, Daniel S.; Spyrou, Vassiliki; Sakkas, Lazaros I.; Rigopoulou, Eirini I.; Bogdanos, Dimitrios P.

    2013-01-01

    p38 mitogen activated protein kinase (p38 MAPK) signaling plays a major role in the modulation of immune-mediated inflammatory responses and therefore has been linked with several autoimmune diseases. The extent of the involvement of p38 MAPK in the pathogenesis of autoimmune blistering diseases has started to emerge, but whether it pays a critical role is a matter of debate. The activity of p38 MAPK has been studied in great detail during the loss of keratinocyte cell-cell adhesions and the development of pemphigus vulgaris (PV) and pemphigus foliaceus (PF). These diseases are characterised by autoantibodies targeting desmogleins (Dsg). Whether autoantibody-antigen interactions can trigger signaling pathways (such as p38 MAPK) that are tightly linked to the secretion of inflammatory mediators which may perpetuate inflammation and tissue damage in pemphigus remains unclear. Yet, the ability of p38 MAPK inhibitors to block activation of the proapoptotic proteinase caspase-3 suggests that the induction of apoptosis may be a consequence of p38 MAPK activation during acantholysis in PV. This review discusses the current evidence for the role of p38 MAPK in the pathogenesis of pemphigus. We will also present data relating to the targeting of these cascades as a means of therapeutic intervention. PMID:23936634

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

    PubMed

    Özcan, Zeynep; Gül, Gizem; Yaman, Ibrahim

    2015-08-01

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

  16. Lobolide, a diterpene, blockades the NF-κB pathway and p38 and ERK MAPK activity in macrophages in vitro

    PubMed Central

    Lv, Xiao-fen; Chen, Si-han; Li, Jie; Fang, Jian-ping; Guo, Yue-wei; Ding, Kan

    2012-01-01

    Aim: Recent studies have shown that constitutive activation of the nuclear factor κB (NF-κB) plays a key role in chronic inflammation and cancers. The aim of this study was to characterize lobolide, a cembrane diterpene, as a drug candidate targeting the NF-κB signaling pathway. Methods: A HEK 293/NF-κB-Luc stable cell line was constructed to evaluate the effect of lobolide on NF-κB activation. THP-1 human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were tested. Lipopolysaccharide (LPS)-induced TNFα and IL-1β production and activation of the TAK1-IKK-NF-κB pathway were studied using ELISA and Western blot analysis. Results: In HEK 293/NF-κB-Luc stable cells, lobolide (0.19–50 μmol/L) inhibited NF-κB activation in a concentration-dependent manner with an IC50 value of 4.2±0.3 μmol/L. Treatment with lobolide (2.5–10 μmol/L) significantly suppressed LPS-induced production of TNFα and IL-1β in both THP-1 cells and PBMCs. In THP-1 cells, the suppression was partially caused by blockade of the translocation of NF-κB from the cytoplasm to the nucleus via affecting the TAK1-IKK-NF-κB pathway and p38 and ERK MAPK activity. Conclusion: Lobolide is a potential inhibitor of the NF-κB pathway, which blocks the translocation of NF-κB from the cytoplasm to the nucleus. Lobolide inhibits LPS-stimulated TNFα and IL-1β release, suggesting that the compound might be an anti-inflammatory compound. PMID:22922340

  17. Molecular characterization of a Fus3/Kss1 type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1.

    PubMed

    Guo, Jun; Dai, Xiwei; Xu, Jin-Rong; Wang, Yulin; Bai, Pengfei; Liu, Furong; Duan, Yinghui; Zhang, Hong; Huang, Lili; Kang, Zhensheng

    2011-01-01

    Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes the destructive wheat stripe rust disease worldwide. Due to the lack of reliable transformation and gene disruption method, knowledge about the function of Pst genes involved in pathogenesis is limited. Mitogen-activated protein kinase (MAPK) genes have been shown in a number of plant pathogenic fungi to play critical roles in regulating various infection processes. In the present study, we identified and characterized the first MAPK gene PsMAPK1 in Pst. Phylogenetic analysis indicated that PsMAPK1 is a YERK1 MAP kinase belonging to the Fus3/Kss1 class. Single nucleotide polymerphisms (SNPs) and insertion/deletion were detected in the coding region of PsMAPK1 among six Pst isolates. Real-time RT-PCR analyses revealed that PsMAPK1 expression was induced at early infection stages and peaked during haustorium formation. When expressed in Fusarium graminearum, PsMAPK1 partially rescued the map1 mutant in vegetative growth and pathogenicity. It also partially complemented the defects of the Magnaporthe oryzae pmk1 mutant in appressorium formation and plant infection. These results suggest that F. graminearum and M. oryzae can be used as surrogate systems for functional analysis of well-conserved Pst genes and PsMAPK1 may play a role in the regulation of plant penetration and infectious growth in Pst. PMID:21779350

  18. Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-κB Signaling Pathway

    PubMed Central

    Chou, Ruey-Hwang; Hsieh, Shu-Ching; Yu, Yung-Luen; Huang, Min-Hsien; Huang, Yi-Chang; Hsieh, Yi-Hsien

    2013-01-01

    Fisetin (3,3’,4’,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion. PMID:23940799

  19. Fisetin inhibits migration and invasion of human cervical cancer cells by down-regulating urokinase plasminogen activator expression through suppressing the p38 MAPK-dependent NF-κB signaling pathway.

    PubMed

    Chou, Ruey-Hwang; Hsieh, Shu-Ching; Yu, Yung-Luen; Huang, Min-Hsien; Huang, Yi-Chang; Hsieh, Yi-Hsien

    2013-01-01

    Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion.

  20. Berberine attenuates high glucose-induced fibrosis by activating the G protein-coupled bile acid receptor TGR5 and repressing the S1P2/MAPK signaling pathway in glomerular mesangial cells.

    PubMed

    Yang, Zhiying; Li, Jie; Xiong, Fengxiao; Huang, Junying; Chen, Cheng; Liu, Peiqing; Huang, Heqing

    2016-08-15

    Berberine (BBR) exerts powerful renoprotective effects on diabetic nephropathy (DN), but the underlying mechanisms remain unclear. We previously demonstrated that activation of the G protein-coupled bile acid receptor TGR5 ameliorates diabetic nephropathy by inhibiting the activation of the sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 2 (S1P2) signaling pathway. In this study, we explored the role of TGR5 in the BBR-induced downregulation of sphingosine 1-phosphate receptor 2 (S1P2)/mitogen-activated protein kinase (MAPK)-mediated fibrosis in glomerular mesangial cells (GMCs). Results showed that, BBR suppressed the expression of FN, ICAM-1, and TGF-β1 in high-glucose cultures of GMCs, and the phosphorylation level of c-Jun/c-Fos was downregulated. The high glucose lowered TGR5 expression in a time-dependent manner; this effect was reversed by BBR in a dose-dependent manner. The TGR5 agonist INT-777 decreased the high glucose-induced FN, ICAM-1, and TGF-β1 protein contents. In addition, TGR5 siRNA blocked S1P2 degradation by BBR. And MAPK signaling, which plays important regulatory roles in the pathological progression of DN, was activated by TGR5 siRNA. Apart from this, MAPK signaling as well as FN, ICAM-1, and TGF-β1 suppressed by BBR under high glucose conditions were limited by TGR5 depletion. Thus, BBR decreases FN, ICAM-1, and TGF-β1 levels under high glucose conditions in GMCs possibly by activating TGR5 and inhibiting S1P2/MAPK signaling. PMID:27292312

  1. Piperine Plays an Anti-Inflammatory Role in Staphylococcus aureus Endometritis by Inhibiting Activation of NF-κB and MAPK Pathways in Mice.

    PubMed

    Zhai, Wen-Jun; Zhang, Zhen-Biao; Xu, Nian-Nian; Guo, Ying-Fang; Qiu, Changwei; Li, Cheng-Ye; Deng, Gan-Zhen; Guo, Meng-Yao

    2016-01-01

    Endometritis is commonly caused by pathogenic microorganisms, including Staphylococcus aureus (S. aureus). Piperine, which is a natural medicine, has shown a variety of biological activities. To explore the effect and mechanism of piperine on S. aureus endometritis, a mouse model of S. aureus endometritis was successfully established in the present study. Histopathological changes were observed with H&E staining, cytokines were analyzed by ELISA, mRNA was analyzed by qPCR, and proteins were detected by western blot. The results showed that piperine could significantly alleviate inflammatory injury in S. aureus endometritis. The qPCR and ELISA results showed that piperine effectively reduced the S. aureus-induced overexpression of TNF-α, IL-1β, and IL-6 but increased the expression of IL-10. The S. aureus-induced inflammation was related to TLR-2 and TLR-4 because the results showed that their expression was increased in S. aureus infection but then decreased with piperine treatment. To further confirm that piperine caused an anti-inflammatory response by targeting NF-κB and MAPKs, the expression of I-κB, p65, p38, ERK, and JNK was measured. The phosphorylation of I-κB, p65, p38, ERK, and JNK was inhibited by piperine in a dose-dependent manner. All of the results indicated that piperine may be a potential anti-inflammatory drug both in endometritis and in other S. aureus-induced diseases. PMID:27293467

  2. Piperine Plays an Anti-Inflammatory Role in Staphylococcus aureus Endometritis by Inhibiting Activation of NF-κB and MAPK Pathways in Mice

    PubMed Central

    Zhai, Wen-jun; Zhang, Zhen-biao; Xu, Nian-nian; Guo, Ying-fang; Qiu, Changwei; Li, Cheng-ye; Deng, Gan-zhen; Guo, Meng-yao

    2016-01-01

    Endometritis is commonly caused by pathogenic microorganisms, including Staphylococcus aureus (S. aureus). Piperine, which is a natural medicine, has shown a variety of biological activities. To explore the effect and mechanism of piperine on S. aureus endometritis, a mouse model of S. aureus endometritis was successfully established in the present study. Histopathological changes were observed with H&E staining, cytokines were analyzed by ELISA, mRNA was analyzed by qPCR, and proteins were detected by western blot. The results showed that piperine could significantly alleviate inflammatory injury in S. aureus endometritis. The qPCR and ELISA results showed that piperine effectively reduced the S. aureus-induced overexpression of TNF-α, IL-1β, and IL-6 but increased the expression of IL-10. The S. aureus-induced inflammation was related to TLR-2 and TLR-4 because the results showed that their expression was increased in S. aureus infection but then decreased with piperine treatment. To further confirm that piperine caused an anti-inflammatory response by targeting NF-κB and MAPKs, the expression of I-κB, p65, p38, ERK, and JNK was measured. The phosphorylation of I-κB, p65, p38, ERK, and JNK was inhibited by piperine in a dose-dependent manner. All of the results indicated that piperine may be a potential anti-inflammatory drug both in endometritis and in other S. aureus-induced diseases. PMID:27293467

  3. Regulatory Roles of MAPK Phosphatases in Cancer

    PubMed Central

    Low, Heng Boon

    2016-01-01

    The mitogen-activated protein kinases (MAPKs) are key regulators of cell growth and survival in physiological and pathological processes. Aberrant MAPK signaling plays a critical role in the development and progression of human cancer, as well as in determining responses to cancer treatment. The MAPK phosphatases (MKPs), also known as dual-specificity phosphatases (DUSPs), are a family of proteins that function as major negative regulators of MAPK activities in mammalian cells. Studies using mice deficient in specific MKPs including MKP1/DUSP1, PAC-1/DUSP2, MKP2/DUSP4, MKP5/DUSP10 and MKP7/DUSP16 demonstrated that these molecules are important not only for both innate and adaptive immune responses, but also for metabolic homeostasis. In addition, the consequences of the gain or loss of function of the MKPs in normal and malignant tissues have highlighted the importance of these phosphatases in the pathogenesis of cancers. The involvement of the MKPs in resistance to cancer therapy has also gained prominence, making the MKPs a potential target for anti-cancer therapy. This review will summarize the current knowledge of the MKPs in cancer development, progression and treatment outcomes. PMID:27162525

  4. Molecular Cloning of MAPK Gene Family Using Synthetic Oligonucleotide Probe.

    PubMed

    Zhou, Song; Wang, Qin; Chen, Jing; Chen, Jiang-Ye

    1999-01-01

    MAPK(mitogen activated protein kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role in several different signal transduction pathways. The MAPKs may also have a role in morphorgenesis of Candida albicans. An oligonucleotide probe was used to screen novel MAPKs in C. albicans. All MAPKs shared high homogeneity in their eleven kinase subdomains, especially subdoman VII and VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and the two known MAPKs in C. albicans CEK1 and MKC1 have only one different nucleotide in that DNA sequence. This probe was hybridized with C. albicans genomic DNA. Under stringent conditions, the probe could only hybridize with CEK1 and MKC1 gene fragment. But when hybridized at 40 degrees in non-SDS solution, two novel bands appeared. This condition was used to screen SC5314 DNA library, and many positive clones with different hybridization density were obtained. The strongest hybridization clones were identified to contain CEK1 and MKC1 gene. From the stronger positive hybridization clones, two novel genes were identified. The first gene, named CRK1(CDC2-related protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is closest to SGV1 from S. cerevisiae (with homology 47%) and PITALRE from human (with homology 41%), both of which are CDC2-related protein kinases. The second gene called CEK2(Candida albicans extracelluar signal-regulated kinase 2) is a novel MAPK of Candida albicans, which shares the highest identity with CEK1 and its S. cerevisiae homologs, FUS3 and KSS1, two redundant MAPKs in yeast pheromone response and morphogenesis. PMID:12114967

  5. The Asian-American variant of human papillomavirus type 16 exhibits higher activation of MAPK and PI3K/AKT signaling pathways, transformation, migration and invasion of primary human keratinocytes.

    PubMed

    Hochmann, Jimena; Sobrinho, João S; Villa, Luisa L; Sichero, Laura

    2016-05-01

    Asian-American (AA) HPV-16 variants are associated with higher risk of cancer. Abnormal activation of intracellular signaling play a critical role in cancer development and progression. Our aim was to elucidate mechanisms underlying the higher oncogenic potential attributed to AA variant. We evaluated activation of MAPK and PI3K/AKT pathways in primary human keratinocytes (PHKs) transduced with E6/E7 of three HPV-16 variants: E-P, AA, E-350G. Phenotypes examined included migration, anchorage independent growth and invasion. AA PHKs presented the highest levels of active proteins involved in all cascades analyzed: MAPK-ERK, MAPK-p38 and PI3K-AKT. AA PHKs were more efficient in promoting anchorage independent growth, and in stimulating cell migration and invasion. MEK1 inhibition decreased migration. The mesenchymal phenotype marker vimentin was increased in AA PHKs. Our results suggest that MEK1, ERK2, AKT2 hyperactivation influence cellular behavior by means of GSK-3b inactivation and EMT induction prompting AA immortalized PHKs to more efficiently surpass carcinogenesis steps.

  6. Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma.

    PubMed

    Yang, J; He, J; Wang, J; Cao, Y; Ling, J; Qian, J; Lu, Y; Li, H; Zheng, Y; Lan, Y; Hong, S; Matthews, J; Starbuck, M W; Navone, N M; Orlowski, R Z; Lin, P; Kwak, L W; Yi, Q

    2012-09-01

    Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism-activation of p38 signaling in myeloma cells-by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease. PMID:22425892

  7. Age-dependent changes of the antioxidant system in rat livers are accompanied by altered MAPK activation and a decline in motor signaling

    PubMed Central

    Yang, Wei; Burkhardt, Britta; Fischer, Luise; Beirow, Maja; Bork, Nadja; Wönne, Eva C.; Wagner, Cornelia; Husen, Bettina; Zeilinger, Katrin; Liu, Liegang; Nussler, Andreas K.

    2015-01-01

    Aging is characterized by a progressive decrease of cellular functions, because cells gradually lose their capacity to respond to injury. Increased oxidative stress is considered to be one of the major contributors to age-related changes in all organs including the liver. Our study has focused on elucidating whether important antioxidative enzymes, the mTOR pathway, and MAPKs exhibit age-dependent changes in the liver of rats during aging. We found an age-dependent increase of GSH in the cytosol and mitochondria. The aged liver showed an increased SOD enzyme activity, while the CAT enzyme activity decreased. HO-1 and NOS-2 gene expression was lower in adult rats, but up-regulated in aged rats. Western blot analysis revealed that SOD1, SOD2, GPx, GR, γ-GCL, and GSS were age-dependent up-regulated, while CAT remained constant. We also demonstrated that the phosphorylation of Akt, JNK, p38, and TSC2Ser1254 decreased while ERK1/2 and TSC2Thr1462 increased age-dependently. Furthermore, our data show that the mTOR pathway seems to be activated in livers of aged rats, and hence stimulating cell proliferation/regeneration, as confirmed by an age-dependent increase of PCNA and p-eIF4ESer209 protein expression. Our data may help to explain the fact that liver cells only proliferate in cases of necessity, like injury and damage. In summary, we have demonstrated that, age-dependent changes of the antioxidant system and stress-related signaling pathways occur in the livers of rats, which may help to better understand organ aging. PMID:27004051

  8. Transmembrane/cytoplasmic, rather than catalytic, domains of Mmp14 signal to MAPK activation and mammary branching morphogenesis via binding to integrin β1

    PubMed Central

    Mori, Hidetoshi; Lo, Alvin T.; Inman, Jamie L.; Alcaraz, Jordi; Ghajar, Cyrus M.; Mott, Joni D.; Nelson, Celeste M.; Chen, Connie S.; Zhang, Hui; Bascom, Jamie L.; Seiki, Motoharu; Bissell, Mina J.

    2013-01-01

    Epithelial cell invasion through the extracellular matrix (ECM) is a crucial step in branching morphogenesis. The mechanisms by which the mammary epithelium integrates cues from the ECM with intracellular signaling in order to coordinate invasion through the stroma to make the mammary tree are poorly understood. Because the cell membrane-bound matrix metalloproteinase Mmp14 is known to play a key role in cancer cell invasion, we hypothesized that it could also be centrally involved in integrating signals for mammary epithelial cells (MECs) to navigate the collagen 1 (CL-1)-rich stroma of the mammary gland. Expression studies in nulliparous mice that carry a NLS-lacZ transgene downstream of the Mmp14 promoter revealed that Mmp14 is expressed in MECs at the tips of the branches. Using both mammary organoids and 3D organotypic cultures, we show that MMP activity is necessary for invasion through dense CL-1 (3 mg/ml) gels, but dispensable for MEC branching in sparse CL-1 (1 mg/ml) gels. Surprisingly, however, Mmp14 without its catalytic activity was still necessary for branching. Silencing Mmp14 prevented cell invasion through CL-1 and disrupted branching altogether; it also reduced integrin β1 (Itgb1) levels and attenuated MAPK signaling, disrupting Itgb1-dependent invasion/branching within CL-1 gels. FRET imaging revealed that Mmp14 associates directly with Itgb1. We identified a domain of Mmp14 that is required for modulating the levels of Itgb1, MEC signaling and the rate of invasion within CL-1. These results shed light on hitherto undescribed non-proteolytic activities of Mmp14 that are necessary for the Itgb1-dependent biochemical and mechanical signals that regulate branching in the mammary epithelium. PMID:23250208

  9. Activation of the calcium-sensing receptor promotes apoptosis by modulating the JNK/p38 MAPK pathway in focal cerebral ischemia-reperfusion in mice

    PubMed Central

    Zhen, Yilan; Ding, Caijuan; Sun, Jiaqiang; Wang, Yanan; Li, Sheng; Dong, Liuyi

    2016-01-01

    Exact mechanism of cerebral ischemic stroke remains unclear. The calcium-sensing receptor (CaSR), a G-protein coupled receptor, has been reported to participate in the pathology of myocardial ischemia-reperfusion (I/R) injury and myocardial hypertrophy. Nevertheless, only a limited number of studies have been conducted to investigate the role of CaSR in cerebral ischemic stroke. This study was to investigate the effect of CaSR activation on cerebral ischemic stroke. Male adult Kunming mice were subjected to 2-h focal cerebral ischemia followed by 22-h reperfusion. Then, the brain was collected, and the expression of CaSR, JNK, p38, Bcl-2, and Bax was detected by Western blot assay. The morphology of neurons in the brain was evaluated by HE staining. Neurological function was scored, and the infarct volume was determined by TTC (triphenyltetrazolium chloride) staining. Results showed that ischemia/reperfusion (I/R) increased CaSR expression and induced neuronal apoptosis in the brain. Gadolinium trichloride (GdCl3), an agonist of CaSR, further deteriorated neurological dysfunction, increased infarct volume, enhanced CaSR expression, and promoted neuronal apoptosis. In addition, GdCl3 unregulated expression of Bax, p-JNK, and p-p38, and down-regulated Bcl-2 expression during I/R, which were attenuated by NPS2390, an inhibitor of CaSR. In conclusion, the CaSR activation promotes apoptosis in focal cerebral I/R in mice, which may be related to the activation of JNK/p38 MAPK signalling pathway. Targeting CaSR may be a novel strategy for the prevention and treatment of cerebral ischemic stroke. PMID:27158378

  10. Active Debris Removal: Current Status of Activities in CNES

    NASA Astrophysics Data System (ADS)

    Bonnal, Christophe; Ruault, Jean-Marc; Desjean, Marie-Christine

    2013-08-01

    Most of the ongoing studies led at worldwide level, mainly through IADC Actions, conclude that in order to keep a stable Low Earth Orbit environment in the coming decades, it may be necessary to retrieve some 5 to 10 large objects annually. These operations, known as Active Debris Removal (ADR), raise a huge amount of difficulties in numerous domains: political, legal, insurance, defense, financing and, last but not least, technical questions. The current paper aims at reviewing the current status of the ADR activities led by CNES both at National and Multi-lateral level. The first question which is raised is that of the high level requirements to be applied. What are the requirements coming from the operators; do we want to stabilize the environment, decrease it or could we accept some increase over the years; when do we have to act; can we baseline random reentry of such large objects or do we have to stick to controlled destructive reentries?… There may not yet be clear answers to these points, so efforts at international level are required. The second part of the paper deals with the potential solutions at system level. Numerous possibilities can be identified, depending on the size of the launcher and of the strategy selected to de-orbit the debris. Large space tugs visiting some 10 debris or small dedicated chasers launched as piggyback are among the solutions which have been traded. The currently preferred solution is described in details. The third part of the paper is devoted to the chaser-debris operations themselves, following five key functions; - the long range rendezvous, - the short range rendezvous up to contact, - the mechanical interfacing of the debris, - its control by the chaser, when required, - the de-orbiting maneuver itself. For each of these functions, the current status of available technologies is described, enabling the identification of the most critical ones requiring additional R&T effort and subsequent demonstrations. Among them

  11. Grape Seed Procyanidin Reversal of P-glycoprotein Associated Multi-Drug Resistance via Down-regulation of NF-κB and MAPK/ERK Mediated YB-1 Activity in A2780/T Cells

    PubMed Central

    Wang, Sheng-qi; Duan, Lian; Huo, Qi-lu; Ren, Fei; Li, Guo-feng

    2013-01-01

    The expression and function of P-glycoprotein (P-gp) is associated with the phenotype of multi-drug resistance (MDR), leading chemotherapy failure of patients suffered with cancer. Grape seed procyanidin(GSP) is a natural polyphenol supplement with anti-inflammatory effect. Present study assessed a new use of GSP on the MDR reversal activity and its possible molecular mechanisms in MDR1-overpressing paclitaxel resistant ovarian cancer cells. Our results showed GSP significantly enhanced the cytotoxicity of paclitaxel and adriamycin in paclitaxel resistant A2780/T cells but its parental A2780 cells. Furthermore, GSP strongly inhibited P-gp expression by blocking MDR1 gene transcription, as well as, increased the intracellular accumulation of the P-gp substrate rhodamine-123 in A2780/T cells. Nuclear factor-κB(NF-κB) activity, IκB degradation level and NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS) and receptor activator for nuclear factor-κB ligand (RANKL) were markedly inhibited by pre-treatment with GSP. Meanwhile, GSP inhibited MAPK/ERK pathway by decreasing the phosphorylation of ERK1/2, resulting in reduced the Y-box binding protein 1 (YB-1) activation with blocking its nuclear translocation. Moreover, the up-regulation of P-gp expression, the activation of AKT/NF-κB and MAPK/ERK pathway induced by LPS was attenuated by GSP administration. Compared with PDTC and U1026, inhibitor of NF-κB and MAPK/ERK respectively, GSP showed the same tendency of down-regulating NF-κB and MAPK/ERK mediated YB-1 activities. Thus, GSP reverses P-gp associated MDR by inhibiting the function and expression of P-gp through down-regulation of NF-κB activity and MAPK/ERK pathway mediated YB-1 nuclear translocation, offering insight into the mechanism of reversing MDR by natural polyphenol supplement compounds. GSP could be a new potential MDR reversal agent used for combination therapy with chemotherapeutics in clinic. PMID:23967153

  12. Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

    PubMed Central

    Yang, Jing; He, Jin; Wang, Ji; Cao, Yabing; Ling, Jianhua; Qian, Jianfei; Lu, Yong; Li, Haiyan; Zheng, Yuhuan; Lan, Yongsheng; Hong, Sungyoul; Matthews, Jairo; Starbuck, Michael W; Navone, Nora M; Orlowski, Robert Z.; Lin, Pei; Kwak, Larry W.; Yi, Qing

    2012-01-01

    Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into SCID or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism—sactivation of p38 signaling in myeloma cells—by which myeloma cells induce osteolytic bone lesions and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease. PMID:22425892

  13. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR

    PubMed Central

    Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

    2016-01-01

    Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

  14. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR.

    PubMed

    Du, Qian; Huang, Yong; Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

    2016-04-01

    Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

  15. Indomethacin induces increase in gastric epithelial tight junction permeability via redistribution of occludin and activation of p38 MAPK in MKN-28 Cells.

    PubMed

    Thakre-Nighot, Meghali; Blikslager, Anthony T

    2016-01-01

    Tight Junctions (TJ) create a paracellular barrier that is compromised when nonsteriodal anti-inflammatory drugs (NSAIDs) injure the gastric epithelium, leading to increased permeability. However, the mechanism of NSAID-induced gastric injury is unclear. Here, we examined the effect of indomethacin on barrier function and TJ in gastric MKN-28 cells. In concentration response studies, 500 µm indomethacin induced a significant decrease in transepithelial resistance (TER; 380 vs. 220 Ω·cm(2) for control and indomethacin-treated cells respectively, p < 0.05), and increased dextran permeability by 0.2 vs 1.2 g/l (p < 0.05). These changes in barrier function were completely ameliorated by the p38 MAPK inhibitor (SB-203580) but not by JNK inhibitor (SP-600125) or MEK/ERK inhibitor (PD-98059). SiRNA knock down of p38 MAPK prevented the loss of barrier function caused by indomethacin in MKN-28 cells. Western analyses of TJ proteins revealed that expression of occludin was reduced by indomethacin, whereas there was no change in other TJ proteins. The loss of occludin expression induced by indomethacin was prevented by inhibition of p38 MAPK but not JNK or ERK and also by siRNA of p38 MAPK. Immunofluorescence revealed disruption of occludin localization at the site of the tight junction in indomethacin-treated cells, and this was attenuated by inhibition of p38 MAPK. NSAID injury to murine gastric mucosa on Ussing chambers revealed that indomethacin caused a significant drop in TER and increased paracellular permeability. Pretreatment with the p38 MAPK inhibitor significantly attenuated the disruption of barrier function, but JNK and MEK/ERK inhibition had no effect. Western blot analysis on gastric mucosa reveled loss of TJ protein occludin by indomethacin, which was prevented by inhibition of p38 MAPK. This data suggests that indomethacin compromises the gastric epithelial barrier via p38 MAPK inducing occludin alterations in the TJs. PMID:27583191

  16. A MAPK-Driven Feedback Loop Suppresses Rac Activity to Promote RhoA-Driven Cancer Cell Invasion

    PubMed Central

    Hetmanski, Joseph H. R.; Zindy, Egor; Schwartz, Jean-Marc; Caswell, Patrick T.

    2016-01-01

    Cell migration in 3D microenvironments is fundamental to development, homeostasis and the pathobiology of diseases such as cancer. Rab-coupling protein (RCP) dependent co-trafficking of α5β1 and EGFR1 promotes cancer cell invasion into fibronectin (FN) containing extracellular matrix (ECM), by potentiating EGFR1 signalling at the front of invasive cells. This promotes a switch in RhoGTPase signalling to inhibit Rac1 and activate a RhoA-ROCK-Formin homology domain-containing 3 (FHOD3) pathway and generate filopodial actin-spike protrusions which drive invasion. To further understand the signalling network that drives RCP-driven invasive migration, we generated a Boolean logical model based on existing network pathways/models, where each node can be interrogated by computational simulation. The model predicted an unanticipated feedback loop, whereby Raf/MEK/ERK signalling maintains suppression of Rac1 by inhibiting the Rac-activating Sos1-Eps8-Abi1 complex, allowing RhoA activity to predominate in invasive protrusions. MEK inhibition was sufficient to promote lamellipodia formation and oppose filopodial actin-spike formation, and led to activation of Rac and inactivation of RhoA at the leading edge of cells moving in 3D matrix. Furthermore, MEK inhibition abrogated RCP/α5β1/EGFR1-driven invasive migration. However, upon knockdown of Eps8 (to suppress the Sos1-Abi1-Eps8 complex), MEK inhibition had no effect on RhoGTPase activity and did not oppose invasive migration, suggesting that MEK-ERK signalling suppresses the Rac-activating Sos1-Abi1-Eps8 complex to maintain RhoA activity and promote filopodial actin-spike formation and invasive migration. Our study highlights the predictive potential of mathematical modelling approaches, and demonstrates that a simple intervention (MEK-inhibition) could be of therapeutic benefit in preventing invasive migration and metastasis. PMID:27138333

  17. ERK/MAPK regulates ERRγ expression, transcriptional activity and receptor-mediated tamoxifen resistance in ER+ breast cancer.

    PubMed

    Heckler, Mary M; Thakor, Hemang; Schafer, Cara C; Riggins, Rebecca B

    2014-05-01

    Selective estrogen receptor modulators such as tamoxifen (TAM) significantly improve breast cancer-specific survival for women with estrogen receptor-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. The resistant phenotype is usually not driven by loss or mutation of the estrogen receptor; instead, changes in multiple proliferative and/or survival pathways over-ride the inhibitory effects of TAM. Estrogen-related receptor γ (ERRγ) is an orphan member of the nuclear receptor superfamily that promotes TAM resistance in ER+ breast cancer cells. This study sought to clarify the mechanism(s) by which this orphan nuclear receptor is regulated, and hence affects TAM resistance. mRNA and protein expression/phosphorylation were monitored by RT-PCR and western blotting, respectively. Site-directed mutagenesis was used to disrupt consensus extracellular signal-regulated kinase (ERK) target sites. Cell proliferation and cell-cycle progression were measured by flow cytometric methods. ERRγ transcriptional activity was assessed by dual-luciferase promoter-reporter assays. We show that ERRγ protein levels are affected by the activation state of ERK/mitogen-activated protein kinase, and mutation of consensus ERK target sites impairs ERRγ-driven transcriptional activity and TAM resistance. These findings shed new light on the functional significance of ERRγ in ER+ breast cancer, and are the first to demonstrate a role for kinase regulation of this orphan nuclear receptor.

  18. MAPK Signaling and ERK1/2 bistability in Asthma

    PubMed Central

    Alam, Rafeul; Gorska, Magdalena M.

    2010-01-01

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

  19. Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways.

    PubMed

    Ma, Xiaojun; Liu, Yupeng; Zhang, Yao; Yu, Xiaobing; Wang, Weiming; Zhao, Dewei

    2014-03-01

    Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.

  20. Calcium Signaling Involvement in Cadmium-Induced Astrocyte Cytotoxicity and Cell Death Through Activation of MAPK and PI3K/Akt Signaling Pathways.

    PubMed

    Jiang, Jiao Hua; Ge, Guo; Gao, Kai; Pang, Ying; Chai, Rui Chao; Jia, Xi Hua; Kong, Jin Ge; Yu, Albert Cheung-Hoi

    2015-09-01

    Cadmium (Cd), a highly ubiquitous toxic heavy metal, can contaminate the environment, including agricultural soil, water and air, via industrial runoff and other sources of pollution. Cd accumulated in the body via direct exposure or through the food chain results in neurodegeneration and many other diseases. Previous studies on its toxicity in the central nervous system (CNS) focused mainly on neurons. To obtain a more comprehensive understanding of Cd toxicity for the CNS, we investigated how astrocytes respond to acute and chronic Cd exposure and its toxic molecular mechanisms. When primary cultures of cerebral cortical astrocytes incubated with 1-300 μM CdCl2, morphological changes, LDH release and cell death were observed in a time and dose-dependent manner. Further studies demonstrated that acute and chronic Cd treatment phosphorylated JNK, p38 and Akt to different degrees, while ERK1/2 was only phosphorylated under low doses of Cd (10 μM) exposure. Inhibition of JNK and PI3K/Akt, but not of p38, could partially protect astrocyte from cytotoxicity in chronic and acute Cd exposure. Moreover, Cd also induced a strong calcium signal, while BAPTA, a specific intracellular calcium (Ca(2+)) chelator, prevented Cd-induced intracellular increase of calcium levels in astrocytes; inhibited the Cd-induced activation of ERK1/2, JNK, p38 and Akt; and also significantly reduced astrocyte cell death. All of these results suggested that the Cd-Ca(2+)-MAPK and PI3K/Akt signaling pathways were involved in Cd-induced toxicity in astrocytes. This toxicity involvement indicates that these pathways may be exploited as a target for the prevention of Cd-induced neurodegenerative diseases. PMID:26248512

  1. Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway.

    PubMed

    Cao, Guosheng; Jiang, Nan; Hu, Yang; Zhang, Yuanyuan; Wang, Guangyun; Yin, Mingzhu; Ma, Xiaonan; Zhou, Kecheng; Qi, Jin; Yu, Boyang; Kou, Junping

    2016-01-01

    Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen-glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1-10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1β (IL-1β) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke. PMID:27589720

  2. Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway

    PubMed Central

    Cao, Guosheng; Jiang, Nan; Hu, Yang; Zhang, Yuanyuan; Wang, Guangyun; Yin, Mingzhu; Ma, Xiaonan; Zhou, Kecheng; Qi, Jin; Yu, Boyang; Kou, Junping

    2016-01-01

    Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen–glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1–10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1β (IL-1β) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke. PMID:27589720

  3. Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-κB and MAPK Activation

    PubMed Central

    Woo, Hong-Jung; Kim, Youngchul

    2015-01-01

    Orostachys japonicus is traditionally used as an inflammatory agent. In this report, we investigated the effects of O. japonicus extract on the expression of genes encoding pathogen-recognition receptors (TLR2, TLR4, NOD1, and NOD2) and proinflammatory factors (iNOS, COX-2, and cytokines) in LPS-stimulated PMA-differentiated THP-1 cells and the NF-κB and MAPK pathways. O. japonicus induced toxicity at high concentrations but had no effect at concentrations lower than 25 μg/mL. O. japonicus inhibited LPS-induced TLR4 and NOD2 mRNA levels, suppressed LPS-induced iNOS and COX-2 transcription and translocation, and downregulated LPS-induced proinflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) mRNA levels. In addition, O. japonicus inhibited LPS-induced NF-κB activation and IκBα degradation and suppressed LPS-induced JNK, p38 MAPK, and ERK phosphorylation. Overall, our results demonstrate that the anti-inflammatory effects of O. japonicus are mediated by suppression of NF-κB and MAPK signaling, resulting in reduced TLR4, NOD2, iNOS, and COX-2 expression and inhibition of inflammatory cytokine expression. PMID:25810745

  4. Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Accelerates the Progression of Renal Fibrosis in Lupus Nephritis by Activating SMAD and p38 MAPK in TGF-β1 Signaling Pathway

    PubMed Central

    Liu, Zhiqin; Xue, Leixi; Liu, Zhichun; Huang, Jun; Wen, Jian; Hu, Ji; Bo, Lin; Yang, Ru

    2016-01-01

    This study aim was to explore the effects of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in lupus nephritis and its potential underlying mechanisms. MRL/lpr mice were used for in vivo experiments and human proximal tubular cells (HK2 cells) were used for in vitro experiments. Results showed that MRL/lpr mice treated with vehicle solution or LV-Control shRNA displayed significant proteinuria and severe renal histopathological changes. LV-TWEAK-shRNA treatment reversed these changes and decreased renal expressions of TWEAK, TGF-β1, p-p38 MAPK, p-Smad2, COL-1, and α-SMA proteins. In vitro, hTWEAK treatment upregulated the expressions of TGF-β1, p-p38 MAPK, p-SMAD2, α-SMA, and COL-1 proteins in HK2 cells and downregulated the expressions of E-cadherin protein, which were reversed by cotreatment with anti-TWEAK mAb or SB431542 treatment. These findings suggest that TWEAK may contribute to chronic renal changes and renal fibrosis by activating TGF-β1 signaling pathway, and phosphorylation of Smad2 and p38 MAPK proteins was also involved in this signaling pathway. PMID:27365897

  5. p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    PubMed Central

    Marengo, B; De Ciucis, C G; Ricciarelli, R; Furfaro, A L; Colla, R; Canepa, E; Traverso, N; Marinari, U M; Pronzato, M A; Domenicotti, C

    2013-01-01

    Neuroblastoma (NB) is the second most common solid pediatric tumor and is characterized by clinical and biological heterogeneity, and stage-IV of the disease represents 50% of all cases. Considering the limited success of present chemotherapy treatment, it has become necessary to find new and effective therapies. In this context, our approach consists of identifying and targeting key molecular pathways associated with NB chemoresistance. This study has been carried out on three stage-IV NB cell lines with different status of MYCN amplification. Cells were exposed to a standard chemotherapy agent, namely etoposide, either alone or in combination with particular drugs, which target intracellular signaling pathways. Etoposide alone induced a concentration-dependent reduction of cell viability and, at very high doses, totally counteracted cell tumorigenicity and neurosphere formation. In addition, etoposide activated p38 mitogen-activated protein kinase (MAPK), AKT and c-Jun N-terminal kinase. Pre-treatment with SB203580, a p38MAPK inhibitor, dramatically sensibilized NB cells to etoposide, strongly reducing the dosage needed to inhibit tumorigenicity and neurosphere formation. Importantly, SB203580–etoposide cotreatment also reduced cell migration and invasion by affecting cyclooxygenase-2, intercellular adhesion molecule-1, C–X–C chemokine receptor-4 and matrix metalloprotease-9. Collectively, our results suggest that p38MAPK inhibition, in combination with standard chemotherapy, could represent an effective strategy to counteract NB resistance in stage-IV patients. PMID:23579276

  6. Social status modulates neural activity in the mentalizing network

    PubMed Central

    Muscatell, Keely A.; Morelli, Sylvia A.; Falk, Emily B.; Way, Baldwin M.; Pfeifer, Jennifer H.; Galinsky, Adam D.; Lieberman, Matthew D.; Dapretto, Mirella; Eisenberger, Naomi I.

    2013-01-01

    The current research explored the neural mechanisms linking social status to perceptions of the social world. Two fMRI studies provide converging evidence that individuals lower in social status are more likely to engage neural circuitry often involved in ‘mentalizing’ or thinking about others' thoughts and feelings. Study 1 found that college students' perception of their social status in the university community was related to neural activity in the mentalizing network (e.g., DMPFC, MPFC, precuneus/PCC) while encoding social information, with lower social status predicting greater neural activity in this network. Study 2 demonstrated that socioeconomic status, an objective indicator of global standing, predicted adolescents' neural activity during the processing of threatening faces, with individuals lower in social status displaying greater activity in the DMPFC, previously associated with mentalizing, and the amygdala, previously associated with emotion/salience processing. These studies demonstrate that social status is fundamentally and neurocognitively linked to how people process and navigate their social worlds. PMID:22289808

  7. Dietary influence on MAPK-signaling pathways and risk of colon and rectal cancer

    PubMed Central

    Slattery, Martha L.; Lundgreen, Abbie; Wolff, Roger K.

    2014-01-01

    Mitogen-activated protein kinase (MAPK) pathways regulate cellular functions including cell proliferation, differentiation, migration, and apoptosis. Associations between genes in the DUSP, ERK1/2, JNK, and p38 MAPK-signaling pathways and dietary factors associated with growth factors, inflammation, and oxidative stress and risk of colon and rectal cancer were evaluated. Data include colon cases (n=1555) and controls (n=1956) and rectal cases (n=754) and controls (n=959). Statistically significant interactions were observed for the MAPK-signaling pathways after adjustment for multiple comparisons. DUSP genes interacted with carbohydrates, mutagen index, calories, calcium, vitamin D, lycopene, dietary fats, folic acid, and selenium. MAPK1, MAPK3, MAPK1 and RAF1 within the ERK1/2 MAPK-signaling pathway interacted with dietary fats and cruciferous vegetables. Within the JNK MAPK-signaling pathway, interactions between MAP3K7 and protein, vitamin C, iron, folic acid, carbohydrates, and cruciferous vegetables; MAP3K10 and folic acid; MAP3K9 and lutein/zeaxanthin; MAPK8 and calcium; MAP3K3 and calcium and lutein; MAP3K1 and cruciferous vegetables. Interaction within the p38-signaling pathway included: MAPK14 with calories, carbohydrates saturated fat, selenium, vitamin C; MAP3K2 and carbohydrates, and folic acid. These data suggest that dietary factors involved in inflammation and oxidative stress interact with MAPK-signaling genes to alter risk of colorectal cancer. PMID:23859041

  8. Inhibition of Raf/MAPK signaling in Xenopus oocyte extracts by Raf-1-specific peptides.

    PubMed

    Radziwill, G; Steinhusen, U; Aitken, A; Moelling, K

    1996-10-01

    Raf-1 is an upstream element of the mitogen-activated protein kinase (MAPK) pathway which leads to cell proliferation and differentiation. In this study Raf-1 derived peptides comprising the conserved amino acid residues Arg89 and Ser259, involved in binding of activated Ras and 14-3-3 proteins, respectively, were shown to interfere with MAPK activation in extracts from immature Xenopus oocytes. Lipids prepared from oocyte extracts can stimulate MAPK in a Ras- and protein kinase C-independent manner. This lipid-induced MAPK activation is blocked by a Raf-1 derived peptide comprising Ser259.

  9. Osmotic Stress, not Aldose Reductase Activity, Directly induces Growth Factors and MAPK Signaling changes during Sugar Cataract Formation

    PubMed Central

    Zhang, Peng; Xing, Kuiyi; Randazzo, James; Blessing, Karen; Lou, Marjorie F.; Kador, Peter F.

    2012-01-01

    In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation. PMID:22710095

  10. Resveratrol and Black Tea Polyphenol Combination Synergistically Suppress Mouse Skin Tumors Growth by Inhibition of Activated MAPKs and p53

    PubMed Central

    George, Jasmine; Singh, Madhulika; Srivastava, Amit Kumar; Bhui, Kulpreet; Roy, Preeti; Chaturvedi, Pranav Kumar; Shukla, Yogeshwer

    2011-01-01

    Cancer chemoprevention by natural dietary agents has received considerable importance because of their cost-effectiveness and wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasing popularity. The present study aims to evaluate the combinatorial chemopreventive effects of resveratrol and black tea polyphenol (BTP) in suppressing two-stage mouse skin carcinogenesis induced by DMBA and TPA. Resveratrol/BTP alone treatment decreased tumor incidence by ∼67% and ∼75%, while combination of both at low doses synergistically decreased tumor incidence even more significantly by ∼89% (p<0.01). This combination also significantly regressed tumor volume and number (p<0.01). Mechanistic studies revealed that this combinatorial inhibition was associated with decreased expression of phosphorylated mitogen-activated protein kinase family proteins: extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, p38 and increased in total p53 and phospho p53 (Ser 15) in skin tissue/tumor. Treatment with combinations of resveratrol and BTP also decreased expression of proliferating cell nuclear antigen in mouse skin tissues/tumors than their solitary treatments as determined by immunohistochemistry. In addition, histological and cell death analysis also confirmed that resveratrol and BTP treatment together inhibits cellular proliferation and markedly induces apoptosis. Taken together, our results for the first time lucidly illustrate that resveratrol and BTP in combination impart better suppressive activity than either of these agents alone and accentuate that development of novel combination therapies/chemoprevention using dietary agents will be more beneficial against cancer. This promising combination should be examined in therapeutic trials of skin and possibly other cancers. PMID:21887248

  11. Cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NF-κB-MT1MMP in activating proMMP-2 by ET-1 in pulmonary artery smooth muscle cells.

    PubMed

    Sarkar, Jaganmay; Chowdhury, Animesh; Chakraborti, Tapati; Chakraborti, Sajal

    2016-04-01

    Treatment of bovine pulmonary artery smooth muscle cells with endothelin-1 (ET-1) caused an increase in the expression and activation of proMMP-2 in the cells. The present study was undertaken to determine the underlying mechanisms involved in this scenario. We demonstrated that (i) pretreatment with NADPH oxidase inhibitor, apocynin; PKC-α inhibitor, Go6976; p(38)MAPK inhibitor SB203580 and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by ET-1; (ii) ET-1 treatment to the cells stimulated NADPH oxidase and PKCα activity, p(38)MAPK phosphorylation as well as NF-κB activation by translocation of NF-κBp65 subunit from cytosol to the nucleus, and subsequently by increasing its DNA-binding activity; (iii) ET-1 increases MT1-MMP expression, which was inhibited upon pretreatment with apocynin, Go6976, SB293580, and Bay 11-7082; (iv) ET-1 treatment to the cells downregulated TIMP-2 level. Although apocynin and Go6976 pretreatment reversed ET-1 effect on TIMP-2 level, yet pretreatment of the cells with SB203580 and Bay 11-7082 did not show any discernible change in TIMP-2 level by ET-1. Overall, our results suggest that ET-1-induced activation of proMMP-2 is mediated via cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NFκB-MT1MMP signaling pathways along with a marked decrease in TIMP-2 expression in the cells. PMID:26910780

  12. Cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NF-κB-MT1MMP in activating proMMP-2 by ET-1 in pulmonary artery smooth muscle cells.

    PubMed

    Sarkar, Jaganmay; Chowdhury, Animesh; Chakraborti, Tapati; Chakraborti, Sajal

    2016-04-01

    Treatment of bovine pulmonary artery smooth muscle cells with endothelin-1 (ET-1) caused an increase in the expression and activation of proMMP-2 in the cells. The present study was undertaken to determine the underlying mechanisms involved in this scenario. We demonstrated that (i) pretreatment with NADPH oxidase inhibitor, apocynin; PKC-α inhibitor, Go6976; p(38)MAPK inhibitor SB203580 and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by ET-1; (ii) ET-1 treatment to the cells stimulated NADPH oxidase and PKCα activity, p(38)MAPK phosphorylation as well as NF-κB activation by translocation of NF-κBp65 subunit from cytosol to the nucleus, and subsequently by increasing its DNA-binding activity; (iii) ET-1 increases MT1-MMP expression, which was inhibited upon pretreatment with apocynin, Go6976, SB293580, and Bay 11-7082; (iv) ET-1 treatment to the cells downregulated TIMP-2 level. Although apocynin and Go6976 pretreatment reversed ET-1 effect on TIMP-2 level, yet pretreatment of the cells with SB203580 and Bay 11-7082 did not show any discernible change in TIMP-2 level by ET-1. Overall, our results suggest that ET-1-induced activation of proMMP-2 is mediated via cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NFκB-MT1MMP signaling pathways along with a marked decrease in TIMP-2 expression in the cells.

  13. Gulf War illnesses are autoimmune illnesses caused by increased activity of the p38/MAPK pathway in CD4+ immune system cells, which was caused by nerve agent prophylaxis and adrenergic load.

    PubMed

    Moss, J I

    2013-12-01

    Sodium chloride intake might increase the risk for the development of autoimmune diseases by increasing the activity of the p38/MAPK pathway in CD4+ cells thereby producing pathogenic TH17 cells which are inflammatory. Two factors (muscarinic and beta adrenergic stimulation), already shown to potentiate each other's toxic effects in whole mice, and have combined amplified sub lethal effects on mouse T cells, can have the same effect on CD4+ signaling pathways as sodium chloride. Sick 1991 Gulf War veterans express elevated Th17 cytokine activity, and therefore may have autoimmune illnesses caused directly by the above mentioned exposures.

  14. Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK

    PubMed Central

    Nagiec, Michal J.; McCarter, Patrick C.; Kelley, Joshua B.; Dixit, Gauri; Elston, Timothy C.; Dohlman, Henrik G.

    2015-01-01

    Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5ND) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or “synthetic,” supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling. PMID:26179917

  15. Global Regulation of a Differentiation MAPK Pathway in Yeast

    PubMed Central

    Chavel, Colin A.; Caccamise, Lauren M.; Li, Boyang; Cullen, Paul J.

    2014-01-01

    Cell differentiation requires different pathways to act in concert to produce a specialized cell type. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth in response to nutrient limitation. Differentiation to the filamentous cell type requires multiple signaling pathways, including a mitogen-activated protein kinase (MAPK) pathway. To identify new regulators of the filamentous growth MAPK pathway, a genetic screen was performed with a collection of 4072 nonessential deletion mutants constructed in the filamentous (Σ1278b) strain background. The screen, in combination with directed gene-deletion analysis, uncovered 97 new regulators of the filamentous growth MAPK pathway comprising 40% of the major regulators of filamentous growth. Functional classification extended known connections to the pathway and identified new connections. One function for the extensive regulatory network was to adjust the activity of the filamentous growth MAPK pathway to the activity of other pathways that regulate the response. In support of this idea, an unregulated filamentous growth MAPK pathway led to an uncoordinated response. Many of the pathways that regulate filamentous growth also regulated each other’s targets, which brings to light an integrated signaling network that regulates the differentiation response. The regulatory network characterized here provides a template for understanding MAPK-dependent differentiation that may extend to other systems, including fungal pathogens and metazoans. PMID:25189875

  16. The IgA1 immune complex-mediated activation of the MAPK/ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy.

    PubMed

    Tamouza, Houda; Chemouny, Jonathan M; Raskova Kafkova, Leona; Berthelot, Laureline; Flamant, Martin; Demion, Marie; Mesnard, Laurent; Paubelle, Etienne; Walker, Francine; Julian, Bruce A; Tissandié, Emilie; Tiwari, Meetu K; Camara, Niels O S; Vrtovsnik, François; Benhamou, Marc; Novak, Jan; Monteiro, Renato C; Moura, Ivan C

    2012-12-01

    IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN.

  17. Ssanghwa-tang, an oriental herbal cocktail, exerts anti-melanogenic activity by suppression of the p38 MAPK and PKA signaling pathways in B16F10 cells

    PubMed Central

    2013-01-01

    Background Ssanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, however, role of SHT on the melanin synthesis has not been checked previously. Thus, the present study was designed to determine the effect of SHT on α-melanocyte stimulating hormone (α-MSH)-induced melanogensis and its mechanisms of action in murine B16F10 melanoma cells. Method Cellular melanin content and tyrosinase activity in murine B16F10 melanoma cells were determined after α-MSH stimulation with or without pre-treatment of SHT at the concentration of 250 and 500 μg/ml. Expression level of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF), and activation of c-AMP-dependent protein kinase (PKA), c-AMP-related element binding protein (CREB), and mitogen-activated protein kinases (MAPKs) were examined by Western blot analysis. Results SHT significantly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of MITF, tyrosinase, and TRP-1. In addition, SHT remarkably suppressed tyrosinase, CRE, and MITF luciferase reporter activity in a resting state as well as in α-MSH-stimulating condition. Phosphorylation of p38 MAPK by α-MSH stimulation was efficiently blocked by SHT pre-treatment. Moreover, SHT as an herbal cocktail showed synergistic anti-melanogenic effect compared with that of each single constituent herb. Conclusion SHT efficiently inhibited c-AMP-induced melanin synthesis in B16F10 cells via suppression of PKA and p38 MAPK signaling pathways and subsequently decreased the level of CREB phosphorylation, MITF, and melanogenic enzymes. These results indicate that SHT may be useful as herbal medicine for treating hyperpigmentation and cosmetics as a skin-whitening agent. PMID:23981281

  18. α-Lipoic Acid Inhibits Expression of IL-8 by Suppressing Activation of MAPK, Jak/Stat, and NF-κB in H. pylori-Infected Gastric Epithelial AGS Cells.

    PubMed

    Choi, Ji Hyun; Cho, Soon Ok; Kim, Hyeyoung

    2016-01-01

    The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. α-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether α-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-κB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without α-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-κB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-κB in AGS cells, which was inhibited by α-lipoic acid. In conclusion, α-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.

  19. α-Lipoic Acid Inhibits Expression of IL-8 by Suppressing Activation of MAPK, Jak/Stat, and NF-κB in H. pylori-Infected Gastric Epithelial AGS Cells

    PubMed Central

    Choi, Ji Hyun; Cho, Soon Ok

    2016-01-01

    The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. α-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether α-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-κB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without α-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-κB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-κB in AGS cells, which was inhibited by α-lipoic acid. In conclusion, α-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation. PMID:26632410

  20. The RASopathies: Developmental syndromes of Ras/MAPK pathway dysregulation

    PubMed Central

    Tidyman, William E.; Rauen, Katherine A.

    2009-01-01

    The Ras/mitogen activated protein kinase (MAPK) pathway is essential in the regulation of the cell cycle, differentiation, growth and cell senescence, all of which are critical to normal development. It is therefore not surprising that its dysregulation has profound effects on development. A class of developmental disorders, the “RASopathies”, is caused by germline mutations in genes that encode protein components of the Ras/MAPK pathway. The vast majority of these mutations result in increased signal transduction down the Ras/MAPK pathway, but usually to a lesser extent than somatic mutations associated with oncogenesis. Each syndrome exhibits unique phenotypic features, however, since they all cause dysregulation of the Ras/MAPK pathway, there are numerous overlapping phenotypic features between the syndromes, including characteristic facial features, cardiac defects, cutaneous abnormalities, neurocognitive delay and a predisposition to malignancies. Here we review the clinical and underlying molecular basis for each of these syndromes. PMID:19467855

  1. Status of EPRI's waterside corrosion activities

    SciTech Connect

    Chexal, V.K.; Torok, R.C. ); Shevde, V.C.; Randall, G.A. ); Horowitz, J.S. )

    1990-01-01

    The Electric Power Research Institute (EPRI) has instituted a program in the area of waterside corrosion. This program is designed to develop methodologies to: identify the specific corrosion problem, and then analyze the situation to help utility personnel develop a cost- effective inspection and remedial action program. To do this, there is an overview program - CHEXPERT - which identifies the types of corrosion which are likely to occur in a given situation. CHEXPERT is an expert system providing advisory and diagnostic services in this area. A number of analytical modules under CHEXPERT have been and are continuing to be developed. These modules are designed to enable utility personnel to perform the quantitative evaluations necessary in order to develop an optimum inspection program. CHEC, CHECMATE, and CHEC-CAV are inspection planning tools for single phase erosion corrosion, two-phase erosion, and cavitation, respectively. CHEC-NDE and CHEC-THK facilitate management of inspection data and assessment of worn component adequacy. CHEC and CHECMATE are currently complete and in wide use; the other modules are at various stages of development. The status, technical basis and use of all the analytical models are presented.

  2. DARPP-32 is required for MAPK/ERK signaling in thyroid cells.

    PubMed

    Chocarro-Calvo, Ana; Zaballos, Miguel A; Santisteban, Pilar; García-Jiménez, Custodia

    2012-03-01

    Modulation of MAPK signaling duration by cAMP defines its physiological output by driving cells toward proliferation or differentiation. Understanding how the kinetics of MAPK signaling are integrated with other cellular signals is a key issue in development and cancer. Here we show that dopamine and cAMP-regulated neuronal phosphoprotein, 32 kDa (DARPP-32), a protein required for thyroid cell differentiation, determines whether MAPK/ERK activation is sustained or transient. Serum, a stimulus that activates MAPK signaling and does not independently increase DARPP-32 levels results in transient activation of the MAPK pathway. By contrast, TSH + (IGF-I) activate MAPK signaling but also independently increase DARPP-32 levels. Our results are consistent with a model in which maintenance of DARPP-32 expression by TSH + IGF-I leads to sustained MAPK signaling. Moreover, the sensitivity of MAPK/ERK signaling in thyroid cells is lost when de novo DARPP-32 expression is blocked by small interfering RNA. Because both DARPP-32 levels and function as inhibitor of protein phosphatase 1, a key inhibitor of MAPK kinase activity, are governed by cAMP/protein kinase A, the results may explain why in thyroid cells cAMP signaling downstream from TSH controls the duration of MAPK pathway activity. Thus, fine-tuning of DARPP-32 levels leads to changes in the kinetics or sensitivity of MAPK/ERK signaling. Given the implications of MAPK signaling in thyroid cancer and the loss of DARPP-32 in tumor and transformed thyroid cells, DARPP-32 may represent a key therapeutic target. PMID:22301787

  3. COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways

    SciTech Connect

    Kook, Sung-Ho; Lim, Shin-Saeng; Cho, Eui-Sic; Lee, Young-Hoon; Han, Seong-Kyu; Lee, Kyung-Yeol; Kwon, Jungkee; Hwang, Jae-Won; Bae, Cheol-Hyeon; Seo, Young-Kwon; Lee, Jeong-Chae

    2014-12-12

    Highlights: • COMP-Ang1 induces Tie-2 activation in BMMSCs, but not in primary osteoblasts. • Tie-2 knockdown inhibits COMP-Ang1-stimulated proliferation and osteoblastogenesis. • Tie-2 knockdown prevents COMP-Ang1-induced activation of PI3K/Akt and p38 MAPK. • COMP-Ang1 induces migration of cells via activation of PI3K/Akt and CXCR4 pathways. • COMP-Ang1 stimulates in vivo migration of PDLSCs into a calvarial defect site of rats. - Abstract: Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

  4. The MAPK cascade in equally cleaving spiralian embryos.

    PubMed

    Lambert, J David; Nagy, Lisa M

    2003-11-15

    Spiralian development is shared by several protostome phyla and characterized by regularities in early cleavage, fate map, and larva. Experimental evidence from multiple spiralian species implicates cells in the D quadrant lineage as the organizer of future axial development of the embryo. However, the mechanisms by which the D quadrant is specified differ between species with equal and unequal spiral cleavage. Equally cleaving mollusc embryos establish the D quadrant via cell-cell interactions between the micromeres and macromeres at the 24- to 36-cell stage. In unequally cleaving embryos, the D quadrant is established at the 4-cell stage via asymmetries in the first 2 cell divisions. We have begun to explore the molecular mechanisms of D quadrant patterning in spiralians. Previously, we showed that, in the unequally cleaving embryo of the mollusc Ilyanassa obsoleta, the MAPK pathway is activated and functionally required in 3D and also in the micromeres known to require a signal from 3D. Here, we examine the role of MAPK signaling in 4 spiralians with equal cleavage. In 3 equally cleaving molluscs, the chiton Chaetopleura, the limpet Tectura, and the snail Lymnaea, the MAPK pathway is activated in the 3D cell but not in the overlying micromeres. In the equally cleaving embryo of the polychaete annelid Hydroides, MAPK activation was not detected in the 3D macromere but was observed in one of its daughter cells, 4d. In addition, inhibiting Tectura MAPK activation disrupts differentiation of 3D and cells induced by it, supporting a functional role for MAPK in axis specification in equally cleaving spiralians. Thus, MAPK signaling may have a conserved role in the D quadrant organizer cell 3D in molluscs. However, there have been at least 2 evolutionary changes in the activation of the MAPK pathway during spiralian evolution. MAPK function in the Ilyanassa micromeres is a recent cooption and, since the divergence of annelids and molluscs, there has been a shift in

  5. p38 MAPK Signaling in Osteoblast Differentiation

    PubMed Central

    Rodríguez-Carballo, Eddie; Gámez, Beatriz; Ventura, Francesc

    2016-01-01

    The skeleton is a highly dynamic tissue whose structure relies on the balance between bone deposition and resorption. This equilibrium, which depends on osteoblast and osteoclast functions, is controlled by multiple factors that can be modulated post-translationally. Some of the modulators are Mitogen-activated kinases (MAPKs), whose role has been studied in vivo and in vitro. p38-MAPK modifies the transactivation ability of some key transcription factors in chondrocytes, osteoblasts and osteoclasts, which affects their differentiation and function. Several commercially available inhibitors have helped to determine p38 action on these processes. Although it is frequently mentioned in the literature, this chemical approach is not always as accurate as it should be. Conditional knockouts are a useful genetic tool that could unravel the role of p38 in shaping the skeleton. In this review, we will summarize the state of the art on p38 activity during osteoblast differentiation and function, and emphasize the triggers of this MAPK. PMID:27200351

  6. Molecular characterization of Helicobacter pylori VacA induction of IL-8 in U937 cells reveals a prominent role for p38MAPK in activating transcription factor-2, cAMP response element binding protein, and NF-kappaB activation.

    PubMed

    Hisatsune, Junzo; Nakayama, Masaaki; Isomoto, Hajime; Kurazono, Hisao; Mukaida, Naofumi; Mukhopadhyay, Asish K; Azuma, Takeshi; Yamaoka, Yoshio; Sap, Jan; Yamasaki, Eiki; Yahiro, Kinnosuke; Moss, Joel; Hirayama, Toshiya

    2008-04-01

    Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IkappaBalpha ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca(2+) entry (SKF96365), and intracellular Ca(2+) channels (dantrolene) blocked VacA-induced IL-8 production. Furthermore, an intracellular Ca(2+) chelator (BAPTA-AM), which inhibited VacA-activated p38 MAPK, caused a dose-dependent reduction in VacA-induced IL-8 secretion by U937 cells, implying a role for intracellular Ca(2+) in mediating activation of MAPK and the canonical NF-kappaB pathway. VacA stimulated translocation of NF-kappaBp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-kappaB pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which is a p38MAPK substrate and binds to the AP-1 site of the IL-8 promoter, inhibited VacA-induced IL-8 production. VacA activated an IL-8 promoter containing an NF-IL-6 site, but not a mutated AP-1 or NF-kappaB site, suggesting direct involvement of the ATF-2/CREB binding region or NF-kappaB-binding regions in VacA-induced IL-8 promoter activation. Thus, in U937 cells, VacA directly increases IL-8 production by activation of the p38 MAPK