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Sample records for mapk activation status

  1. Activation of p38MAPK in microglia after ischemia.

    PubMed

    Walton, K M; DiRocco, R; Bartlett, B A; Koury, E; Marcy, V R; Jarvis, B; Schaefer, E M; Bhat, R V

    1998-04-01

    p38MAPK has been implicated in the regulation of proinflammatory cytokines and apoptosis in vitro. To understand its role in neurodegeneration, we determined the time course and localization of the dually phosphorylated active form of p38MAPK in hippocampus after global forebrain ischemia. Phosphorylated p38MAPK and mitogen-activated protein kinase-activated protein 2 activity increased over 4 days after ischemia. Phosphorylated p38MAPK immunoreactivity was observed in microglia in regions adjacent to, but not in, the dying CA1 neurons. In contrast, neither c-Jun N-terminal kinase 1 nor p42/p44MAPK activity was altered after ischemia. These results provide the first evidence for localization of activated p38MAPK in the CNS and support a role for p38MAPK in the microglial response to stress.

  2. MAPK activation by radio waves

    PubMed Central

    Arthur, J. Simon C.

    2007-01-01

    In this issue of the Biochemical Journal, Freidman et al. report the findings of a study to look at the potential of mobile phones to activate intracellular signalling cascades. They found that radio waves corresponding to the frequency commonly used by mobile phones are able to activate ERK1/2 (extracellular-signal-regulated kinases 1 and 2). This effect was observed even at intensities lower than those emitted by mobile phones that are unable to cause any measurable heating effects. This study provides evidence that radio waves induce ERK1/2 activation downstream of the EGF (epidermal growth factor) receptor, which is in turn activated by the release of reactive oxygen species. PMID:17623008

  3. MAPK activation by radio waves.

    PubMed

    Arthur, J Simon C

    2007-08-01

    In this issue of the Biochemical Journal, Freidman et al. report the findings of a study to look at the potential of mobile phones to activate intracellular signalling cascades. They found that radio waves corresponding to the frequency commonly used by mobile phones are able to activate ERK1/2 (extracellular-signal-regulated kinases 1 and 2). This effect was observed even at intensities lower than those emitted by mobile phones that are unable to cause any measurable heating effects. This study provides evidence that radio waves induce ERK1/2 activation downstream of the EGF (epidermal growth factor) receptor, which is in turn activated by the release of reactive oxygen species.

  4. Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland.

    PubMed

    Ho, A K; Price, D M; Terriff, D; Chik, C L

    2006-06-27

    Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.

  5. The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation

    PubMed Central

    Yurtsever, Zeynep; Scheaffer, Suzanne M.; Romero, Arthur G.; Holtzman, Michael J.; Brett, Tom J.

    2015-01-01

    The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors. PMID:25849390

  6. The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation.

    PubMed

    Yurtsever, Zeynep; Scheaffer, Suzanne M; Romero, Arthur G; Holtzman, Michael J; Brett, Tom J

    2015-04-01

    The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors.

  7. Activation of multiple MAPK pathways (ERKs, JNKs, p38-MAPK) by diverse stimuli in the amphibian heart.

    PubMed

    Aggeli, I K; Gaitanaki, C; Lazou, A; Beis, I

    2001-05-01

    We investigated the expression and activation of three MAPK subfamilies in the isolated perfused amphibian heart. ERK was detected as a 43 kDa band; p38-MAPK was detected as a band corresponding to 38 kDa and JNKs were detected as two bands corresponding to 46 and 52 kDa, respectively. PMA induced the activation of the ERK pathway as assessed by determining the phosphorylation state of ERK and the upstream component MEK1/2. PD98059 abolished this activation. p38-MAPK was phosphorylated by sorbitol (almost 12-fold, maximal within 10-15 min) and JNKs were phosphorylated and activated by sorbitol or anoxia/reoxygenation (approximately 4- and 2.5-fold, respectively). SB203580 completely blocked the activation of p38-MAPK by sorbitol. These results indicate that the MAPK pathways activated by phorbol esters, hyperosmotic stress or anoxia/ reoxygenation in the amphibian heart may have an important role in this experimental system.

  8. 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

  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. Evidence for the Involvement of p38 MAPK Activation in Barnacle Larval Settlement

    PubMed Central

    He, Li-Sheng; Xu, Ying; Matsumura, Kiyotaka; Zhang, Yu; Zhang, Gen; Qi, Shu-Hua; Qian, Pei-Yuan

    2012-01-01

    The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts. PMID:23115639

  11. Chk1 inhibition activates p53 through p38 MAPK in tetraploid cancer cells.

    PubMed

    Vitale, Ilio; Senovilla, Laura; Galluzzi, Lorenzo; Criollo, Alfredo; Vivet, Sonia; Castedo, Maria; Kroemer, Guido

    2008-07-01

    We have previously shown that tetraploid cancer cells succumb through a p53-dependent apoptotic pathway when checkpoint kinase 1 (Chk1) is depleted by small interfering RNAs (siRNAs) or inhibited with 7-hydroxystaurosporine (UCN-01). Here, we demonstrate that Chk1 inhibition results in the activating phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Depletion of p38 MAPK by transfection with a siRNA targeting the alpha isoform of p38 MAPK (p38alpha MAPK) abolishes the phosphorylation of p53 on serines 15 and 46 that is induced by Chk1 knockdown. The siRNA-mediated downregulation and pharmacological inhibition of p38alpha MAPK (with SB 203580) also reduces cell death induced by Chk1 knockdown or UCN-01. These results underscore the role of p38 MAPK as a pro-apoptotic kinase in the p53-dependant pathway for the therapeutic elimination of polyploidy cells.

  12. Quercitrin attenuates osteoporosis in ovariectomized rats by regulating mitogen-activated protein kinase (MAPK) signaling pathways.

    PubMed

    Xing, Li-Zhi; Ni, Huai-Jun; Wang, Yu-Ling

    2017-03-13

    MAPK signaling pathways are crucial in regulating osteogenesis, a genetic disorder affecting the bones. Quercitrin, a type of flavonoid, is widely distributed in nature and involved in many pharmacological activities. But its osteoprotective functions and mechanism in osteoporosis are far from being understood clearly. In this paper, the MAPK upregulation was observed in the ovariectomy-induced bone loss. Quercitrin was found to downregulate MAPK signaling pathways and prevent the ovariectomy-induced deterioration of bone mineral density (BMD), trabecular microstructure, and bone mechanical characteristics. In this study, quercitrin was seen to prevent the progression of the postmenopausal osteoporosis among the rats, which may be mediated by the downregulated MAPK signaling pathways.

  13. 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.

  14. A sestrin-dependent Erk-Jnk-p38 MAPK activation complex inhibits immunity during aging.

    PubMed

    Lanna, Alessio; Gomes, Daniel C O; Muller-Durovic, Bojana; McDonnell, Thomas; Escors, David; Gilroy, Derek W; Lee, Jun Hee; Karin, Michael; Akbar, Arne N

    2017-03-01

    Mitogen-activated protein kinases (MAPKs) including Erk, Jnk and p38 regulate diverse cellular functions and are thought to be controlled by independent upstream activation cascades. Here we show that the sestrins bind to and coordinate simultaneous Erk, Jnk and p38 MAPK activation in T lymphocytes within a new immune-inhibitory complex (sestrin-MAPK activation complex (sMAC)). Whereas sestrin ablation resulted in broad reconstitution of immune function in stressed T cells, inhibition of individual MAPKs allowed only partial functional recovery. T cells from old humans (>65 years old) or mice (16-20 months old) were more likely to form the sMAC, and disruption of this complex restored antigen-specific functional responses in these cells. Correspondingly, sestrin deficiency or simultaneous inhibition of all three MAPKs enhanced vaccine responsiveness in old mice. Thus, disruption of sMAC provides a foundation for rejuvenating immunity during aging.

  15. Identification, nomenclature, and evolutionary relationships of mitogen-activated protein kinase (MAPK) genes in soybean.

    PubMed

    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.

  16. 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

  17. Persistent ERK/MAPK Activation Promotes Lactotrope Differentiation and Diminishes Tumorigenic Phenotype

    PubMed Central

    Booth, Allyson; Trudeau, Tammy; Gomez, Crystal; Lucia, M. Scott

    2014-01-01

    The signaling pathways that govern the lactotrope-specific differentiated phenotype, and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion, are poorly understood. Moreover, the specific role of MAPK signaling in lactotrope proliferation vs differentiation, whether activated phosphorylated MAPK is sufficient for prolactinoma tumor formation remain unknown. Given that oncogenic Ras mutations and persistently activated phosphorylated MAPK are found in human tumors, including prolactinomas and other pituitary tumors, a better understanding of the role of MAPK in lactotrope biology is required. Here we directly examined the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) continuous addition of exogenous epidermal growth factor. We find that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype in vitro and in vivo with GH4 cell xenograft tumors. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only fails to promote cell proliferation, but also diminishes tumorigenic characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation. PMID:25361391

  18. 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.

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

    PubMed

    Huang, Tung-Yi; Lin, Chih-Hung

    2006-10-02

    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.

  20. 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.

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

    DOE PAGES

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

    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

  2. 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.

  3. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

    PubMed Central

    Ng, Mei Ying; Wang, Mei; Casey, Patrick J.; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways. PMID:28166272

  4. 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

  5. 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.

  6. Multiple Bcl-2 family immunomodulators from vaccinia virus regulate MAPK/AP-1 activation.

    PubMed

    Torres, Alice A; Albarnaz, Jonas D; Bonjardim, Cláudio A; Smith, Geoffrey L

    2016-09-01

    Vaccinia virus (VACV) is a poxvirus and encodes many proteins that modify the host cell metabolism or inhibit the host response to infection. For instance, it is known that VACV infection can activate the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway and inhibit activation of the pro-inflammatory transcription factor NF-κB. Since NF-κB and MAPK/AP-1 share common upstream activators we investigated whether six different VACV Bcl-2-like NF-κB inhibitors can also influence MAPK/AP-1 activation. Data presented show that proteins A52, B14 and K7 each contribute to AP-1 activation during VACV infection, and when expressed individually outwith infection. B14 induced the greatest stimulation of AP-1 and further investigation showed B14 activated mainly the MAPKs ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), and their substrate c-Jun (a component of AP-1). These data indicate that the same viral protein can have different effects on distinct signalling pathways, in blocking NF-κB activation whilst leading to MAPK/AP-1 activation.

  7. 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.

  8. Thrombin enhances NGF-mediated neurite extension via increased and sustained activation of p44/42 MAPK and p38 MAPK.

    PubMed

    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.

  9. Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

    PubMed

    Donyo, Maya; Hollander, Dror; Abramovitch, Ziv; Naftelberg, Shiran; Ast, Gil

    2016-04-01

    Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine (PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally, we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion, our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute potential treatments for FD.

  10. TAB3 involves in hepatic insulin resistance through activation of MAPK pathway.

    PubMed

    Zhao, Yun; Tang, Zhuqi; Zhu, Xiaohui; Wang, Xueqin; Wang, Cuifang; Zhang, Wanlu; Xia, Nana; Wang, Suxin; Huang, Jieru; Cui, Shiwei

    2015-12-01

    Insulin resistance is often accompanied by chronic inflammatory responses. The mitogen-activated protein kinase (MAPK) pathway is rapidly activated in response to many inflammatory cytokines. But the functional role of MAPKs in palmitate-induced insulin resistance has yet to be clarified. In this study, we found that transforming growth factor β-activated kinase binding protein-3 (TAB3) was up-regulated in insulin resistance. Considering the relationship between transforming growth factor β-activated kinase (TAK1) and MAPK pathway, we assumed TAB3 involved in insulin resistance through activation of MAPK pathway. To certify this hypothesis, we knocked down TAB3 in palmitate treated HepG2 cells and detected subsequent biological responses. Importantly, TAB3 siRNA directly reversed insulin sensitivity by improving insulin signal transduction. Moreover, silencing of TAB3 could facilitate hepatic glucose uptake, reverse gluconeogenesis and improve ectopic fat accumulation. Meanwhile, we found that the positive effect of knocking down TAB3 was more significant when insulin resistance occurred. All these results indicate that TAB3 acts as a negative regulator in insulin resistance through activation of MAPK pathway.

  11. Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells.

    PubMed

    Klinge, Carolyn M; Blankenship, Kristy A; Risinger, Kelly E; Bhatnagar, Shephali; Noisin, Edouard L; Sumanasekera, Wasana K; Zhao, Lei; Brey, Darren M; Keynton, Robert S

    2005-03-04

    Vascular endothelial cells (EC) are an important target of estrogen action through both the classical genomic (i.e. nuclear-initiated) activities of estrogen receptors alpha and beta (ERalpha and ERbeta) and the rapid "non-genomic" (i.e. membrane-initiated) activation of ER that stimulates intracellular phosphorylation pathways. We tested the hypothesis that the red wine polyphenol trans-resveratrol activates MAPK signaling via rapid ER activation in bovine aortic EC, human umbilical vein EC, and human microvascular EC. We report that bovine aortic EC, human umbilical vein EC, and human microvascular EC express ERalpha and ERbeta. We demonstrate that resveratrol and estradiol (E(2)) rapidly activated MAPK in a MEK-1, Src, matrix metalloproteinase, and epidermal growth factor receptor-dependent manner. Importantly, resveratrol activated MAPK and endothelial nitric-oxide synthase (eNOS) at nm concentrations (i.e. an order of magnitude less than that required for ER genomic activity) and concentrations possibly achieved transiently in serum following oral red wine consumption. Co-treatment with ER antagonists ICI 182,780 or 4-hydroxytamoxifen blocked resveratrol- or E(2)-induced MAPK and eNOS activation, indicating ER dependence. We demonstrate for the first time that ERalpha-and ERbeta-selective agonists propylpyrazole triol and diarylpropionitrile, respectively, stimulate MAPK and eNOS activity. A red but not a white wine extract also activated MAPK, and activity was directly correlated with the resveratrol concentration. These data suggest that ER may play a role in the rapid effects of resveratrol in EC and that some of the atheroprotective effects of resveratrol may be mediated through rapid activation of ER signaling in EC.

  12. Neuroprotective effects of cannabidiol in endotoxin-induced uveitis: critical role of p38 MAPK activation

    PubMed Central

    El-Remessy, A.B.; Tang, Y.; Zhu, G.; Matragoon, S.; Khalifa, Y.; Liu, E.K.; Liu, J-Y.; Hanson, E.; Mian, S.; Fatteh, N.

    2008-01-01

    Purpose Degenerative retinal diseases are characterized by inflammation and microglial activation. The nonpsychoactive cannabinoid, cannabidiol (CBD), is an anti-inflammatory in models of diabetes and glaucoma. However, the cellular and molecular mechanisms are largely unknown. We tested the hypothesis that retinal inflammation and microglia activation are initiated and sustained by oxidative stress and p38 mitogen-activated protein kinase (MAPK) activation, and that CBD reduces inflammation by blocking these processes. Methods Microglial cells were isolated from retinas of newborn rats. Tumor necrosis factor (TNF)-α levels were estimated with ELISA. Nitric oxide (NO) was determined with a NO analyzer. Superoxide anion levels were determined by the chemiluminescence of luminol derivative. Reactive oxygen species (ROS) was estimated by measuring the cellular oxidation products of 2’, 7’-dichlorofluorescin diacetate. Results In retinal microglial cells, treatment with lipopolysaccharide (LPS) induced immediate NADPH oxidase-generated ROS. This was followed by p38 MAPK activation and resulted in a time-dependent increase in TNF-α production. At a later phase, LPS induced NO, ROS, and p38 MAPK activation that peaked at 2-6 h and was accompanied by morphological change of microglia. Treatment with 1 μM CBD inhibited ROS formation and p38 MAPK activation, NO and TNF-α formation, and maintained cell morphology. In addition, LPS-treated rat retinas showed an accumulation of macrophages and activated microglia, significant levels of ROS and nitrotyrosine, activation of p38 MAPK, and neuronal apoptosis. These effects were blocked by treatment with 5 mg/kg CBD. Conclusions Retinal inflammation and degeneration in uveitis are caused by oxidative stress. CBD exerts anti-inflammatory and neuroprotective effects by a mechanism that involves blocking oxidative stress and activation of p38 MAPK and microglia. PMID:19052649

  13. 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.

  14. PDGF-BB-mediated activation of p42(MAPK) is independent of PDGF beta-receptor tyrosine phosphorylation.

    PubMed

    Cartel, N J; Liu, J; Wang, J; Post, M

    2001-10-01

    Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42(MAPK) activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF beta-receptor (PDGFR-beta) did not abrogate PDGF-BB-induced p42(MAPK) activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42(MAPK) activation nor did a construct expressing PDGFR-beta with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-beta lacking the cytoplasmic signaling domain abrogated p42(MAPK) activity. These results suggest that PDGF-BB-mediated activation of p42(MAPK) requires the PDGFR-beta but is independent of its tyrosine phosphorylation.

  15. 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

  16. The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke.

    PubMed

    Sun, Jing; Nan, Guangxian

    2016-05-01

    Protein kinases are critical modulators of a variety of intracellular and extracellular signal transduction pathways, and abnormal phosphorylation events can contribute to disease progression in a variety of diseases. As a result, protein kinases have emerged as important new drug targets for small molecule therapeutics. The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the cell membrane to the nucleus in response to a variety of different stimuli. Because this pathway controls a broad spectrum of cellular processes, including growth, inflammation, and stress responses, it is accepted as a therapeutic target for cancer and peripheral inflammatory disorders. There is also increasing evidence that MAPK is an important regulator of ischemic and hemorrhagic cerebral vascular disease, raising the possibility that it might be a drug discovery target for stroke. In this review, we discuss the MAPK signaling pathway in association with its activation in stroke-induced brain injury.

  17. RAS-MAPK pathway epigenetic activation in cancer: miRNAs in action

    PubMed Central

    Masliah-Planchon, Julien; Garinet, Simon; Pasmant, Eric

    2016-01-01

    The highly conserved RAS-mitogen activated protein kinase (MAPK) signaling pathway is involved in a wide range of cellular processes including differentiation, proliferation, and survival. Somatic mutations in genes encoding RAS-MAPK components frequently occur in many tumors, making the RAS-MAPK a critical pathway in human cancer. Since the pioneering study reporting that let-7 miRNA acted as tumor suppressor by repressing the RAS oncogene, growing evidence has suggested the importance of miRNAs targeting the RAS-MAPK in oncogenesis. MiRNAs alterations in human cancers may act as a rheostat of the oncogenic RAS signal that is often amplified as cancers progress. However, specific mechanisms leading to miRNAs deregulation and their functional consequences in cancer are far from being fully elucidated. In this review, we provide an experimental-validated map of RAS-MAPK oncomiRs and tumor suppressor miRNAs from transmembrane receptor to downstream ERK proteins. MiRNAs could be further considered as potential genetic biomarkers for diagnosis, prognosis, or therapeutic purpose. PMID:26646588

  18. 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.

  19. 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

  20. Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

    PubMed Central

    Vitucci, Mark; Karpinich, Natalie O.; Bash, Ryan E.; Werneke, Andrea M.; Schmid, Ralf S.; White, Kristen K.; McNeill, Robert S.; Huff, Byron; Wang, Sophie; Van Dyke, Terry; Miller, C. Ryan

    2013-01-01

    Background Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies. Methods Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. Results Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. Conclusions These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies. PMID:23814263

  1. 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

  2. The p38 mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis

    PubMed Central

    Schett, G; Zwerina, J; Firestein, G

    2009-01-01

    Chronic inflammatory processes are based on a sustained and tightly regulated communication network among different cells types. This network comprises extracellular mediators such as cytokines, chemokines and matrix-degrading proteases, which orchestrate the participation of cells in the chronic inflammatory process. The mirrors of this outside communication world are intracellular transcription factor pathways, which shuttle information about inflammatory stimuli to the cell nucleus. This review examines the function of one key signal transduction pathway of inflammation—the p38 mitogen-activated protein kinases (p38MAPK). The signalling pathway is considered as crucial for the induction and maintenance of chronic inflammation, and its components thus emerge as interesting molecular targets of small molecule inhibitors for controlling inflammation. This review not only summarises the current knowledge of activation, regulation and function of the p38MAPK pathway but also examines the role of this pathway in clinical disease. It gives an overview of current evidence of p38MAPK activation in inflammatory arthritis and elaborates the key molecular determinants which contribute to p38MAPK activation in joint disease. PMID:17827184

  3. 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…

  4. Differential contribution of hypothalamic MAPK activity to anxiety-like behaviour in virgin and lactating rats.

    PubMed

    Jurek, Benjamin; Slattery, David A; Maloumby, Rodrigue; Hillerer, Katharina; Koszinowski, Sophie; Neumann, Inga D; van den Burg, Erwin H

    2012-01-01

    The c-Raf - MEK1/2 - ERK1/2 mitogen-activated protein kinase (MAPK) intracellular signalling cascade in neurons plays important roles in the control of a variety of behaviours, including social behaviours and anxiety. These roles partially overlap with those described for oxytocin (OXT), and it has been shown that OXT activates the MAPK pathway in the hypothalamus (of male), and hippocampus (of female) rats. Here, by combining behavioural (light/dark box) and biochemical analyses (western blotting), we tested two hypotheses: (i) that OXT is anxiolytic within the hypothalamus of females, and (ii) that this effect, as well as that of lactation-associated anxiolysis, depends on the recruitment of the MAPK pathway. We found that, when injected bilaterally into the hypothalamic paraventricular nucleus (PVN), OXT decreased anxiety-like behaviour in virgins, and that this effect depended on phosphorylation of MEK1/2. MAPK pathway activation in lactation was evident by high phosphorylated (p) MEK1/2 levels, and nuclear translocation of ERK1. The high pMEK1/2 levels were necessary for the anxiolytic phenotype typically observed during lactation. Interestingly, exogenous OXT in lactating rats reduced pMEK1/2 levels without a concomitant effect on anxiety, indicating that OXT receptor activation can lead to recruitment of additional intracellular pathways to modulate MEK activity. Still other pathways could include MEK, but without subsequent activation of ERK, as we did not observe any increase in OXT-induced ERK phosphorylation. Together the results demonstrate that the MAPK pathway, especially MEK1/2, is critically involved in the regulation of anxiety-like behaviour in female rats.

  5. TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway

    PubMed Central

    Marques-Fernandez, F; Planells-Ferrer, L; Gozzelino, R; Galenkamp, K MO; Reix, S; Llecha-Cano, N; Lopez-Soriano, J; Yuste, V J; Moubarak, R S; Comella, J X

    2013-01-01

    Activation of tumor necrosis factor receptor-1 can trigger survival or apoptosis pathways. In many cellular models, including the neuronal cell model PC12, it has been demonstrated that inhibition of protein synthesis is sufficient to render cells sensitive to apoptosis induced by TNFα. The survival effect is linked to the translocation of the transcription factor nuclear factor-kappa B (NF-κB) to the nucleus and activation of survival-related genes such as FLICE-like inhibitory protein long form (FLIP-L) or IAPs. Nonetheless, we previously reported an NF-κB-independent contribution of Bcl-xL to cell survival after TNFα treatment. Here, we demonstrate that NF-κB-induced increase in FLIP-L expression levels is essential for mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) activation. We demonstrate that FLIP-L behaves as a Raf-1 activator through both protein–protein interaction and Raf-1 kinase activation, without the requirement of the classical Ras activation. Importantly, prevention of FLIP-L increase by NF-κB inhibition or knockdown of endogenous FLIP-L blocks MAPK/ERK activation after TNFα treatment. From a functional point of view, we show that inhibition of the MAPK/ERK pathway and the NF-κB pathway are equally relevant to render PC12 cells sensitive to cell death induced by TNFα. Apoptosis induced by TNFα under these conditions is dependent on jun nuclear kinase1/2 JNK1/2-dependent Bim upregulation. Therefore, we report a previously undescribed and essential role for MAPK/ERK activation by FLIP-L in the decision between cell survival and apoptosis upon TNFα stimulation. PMID:23412386

  6. 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

  7. MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia

    PubMed Central

    Gorog, Diana A.; Jabr, Rita I; Tanno, Masaya; Sarafraz, Negin; Clark, James E.; Fisher, Simon G.; Cao, Xou Bin; Bellahcene, Mohamed; Dighe, Kushal; Kabir, Alamgir M. N.; Quinlan, Roy A.; Kato, Kanefusa; Gaestel, Matthias; Marber, Michael S.

    2009-01-01

    MAPKAPK-2 (MK2) is a protein kinase activated downstream of p38-MAPK which phosphorylates the small heat shock proteins HSP27 and αB crystallin and modulates p38-MAPK cellular distribution. p38-MAPK activation is thought to contribute to myocardial ischemic injury; therefore, we investigated MK2 effects on ischemic injury and p38 cellular localization using MK2-deficient mice (KO). Immunoblotting of extracts from Langendorff-perfused hearts subjected to aerobic perfusion or global ischemia or reperfusion showed that the total and phosphorylated p38 levels were significantly lower in MK2−/− compared to MK2+/+ hearts at baseline, but the ratio of phosphorylated/total p38 was similar. These results were confirmed by cellular fractionation and immunoblotting for both cytosolic and nuclear compartments. Furthermore, HSP27 and αB crsytallin phosphorylation were reduced to baseline in MK2−/− hearts. On semiquantitative immunofluorescence laser confocal microscopy of hearts during aerobic perfusion, the mean total p38 fluorescence was significantly higher in the nuclear compared to extranuclear (cytoplasmic, sarcomeric, and sarcolemmal compartments) in MK2+/+ hearts. However, although the increase in phosphorylated p38 fluorescence intensity in all compartments following ischemia in MK2+/+ hearts was lost in MK2−/− hearts, it was basally elevated in nuclei of MK2−/− hearts and was similar to that seen during ischemia in MK2+/+ hearts. Despite these differences, similar infarct volumes were recorded in wild-type MK2+/+ and MK2−/− hearts, which were decreased by the p38 inhibitor SB203580 (1 μM) in both genotypes. In conclusion, p38 MAPK-induced myocardial ischemic injury is not modulated by MK2. However, the absence of MK2 perturbs the cellular distribution of p38. The preserved nuclear distribution of active p38 MAPK in MK2−/− hearts and the conserved response to SB203580 suggests that activation of p38 MAPK may contribute to injury

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

    PubMed

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

    2011-11-04

    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.

  9. WNT3 Inhibits Cerebellar Granule Neuron Progenitor Proliferation and Medulloblastoma Formation via MAPK Activation

    PubMed Central

    Ayrault, Olivier; Kim, Jee Hae; Zhu, Xiaodong; Murphy, David A.; Van Aelst, Linda; Roussel, Martine F.; Hatten, Mary E.

    2013-01-01

    During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical β-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors. PMID:24303070

  10. 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.

  11. Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle.

    PubMed

    Wong, J A; Gosmanov, A R; Schneider, E G; Thomason, D B

    2001-08-01

    Na(+)-K(+)-Cl(-) cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive (86)Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na(+)-K(+)-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na(+)-K(+)-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

  12. Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway.

    PubMed

    Shefer, G; Oron, U; Irintchev, A; Wernig, A; Halevy, O

    2001-04-01

    Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

  13. Alpha(1)- and beta-adrenoceptor stimulation differentially activate p38-MAPK and atrial natriuretic peptide production in the perfused amphibian heart.

    PubMed

    Aggeli, Ioanna-Katerina S; Gaitanaki, Catherine; Lazou, Antigone; Beis, Isidoros

    2002-08-01

    We investigated the activation of p38-MAPK by various adrenergic agents in the perfused Rana ridibunda heart. Phenylephrine (50 micromol l(-1)) rapidly induced the differential activation of all three mitogen-activated protein kinase (MAPK) subfamilies (ERK, JNKs and p38-MAPK) in this experimental system. Focusing on p38-MAPK response to phenylephrine, we found that the kinase phosphorylation reached maximal values at 30 s, declining thereafter to basal values at 15 min. p38-MAPK activation by phenylephrine was verified as exclusively alpha(1)-AR-mediated. Furthermore, SB203580 (1 micromol l(-1)) abolished the kinase phosphorylation by phenylephrine. Isoproterenol (50 micromol l(-1)) was also shown to activate p38-MAPK in a time- and temperature-dependent manner. A marked, sustained p38-MAPK activation profile was observed at 25 degrees C, while at 18 degrees C the kinase response to isoproterenol was modest. Isoproterenol effect on p38-MAPK stimulation was beta-AR-mediated. Immunohistochemical studies revealed the enhanced presence of phosphorylated p38-MAPK and atrial natriuretic peptide (ANP) in both phenylephrine- and isoproterenol-stimulated hearts, a reaction completely blocked by the respective specific antagonists, or the specific p38-MAPK inhibitor SB203580. These findings indicate a functional correlation between p38-MAPK activation and ANP accumulation in the perfused amphibian heart.

  14. 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.

  15. Activation of PPARγ suppresses proliferation and induces apoptosis of esophageal cancer cells by inhibiting TLR4-dependent MAPK pathway.

    PubMed

    Wu, Kai; Yang, Yang; Liu, Donglei; Qi, Yu; Zhang, Chunyang; Zhao, Jia; Zhao, Song

    2016-07-12

    Although substantial studies on peroxisome proliferator-activated receptor g (PPARg) have focused on the mechanisms by which PPARg regulates glucose and lipid metabolism, recent reports have suggested that PPARg shows tumorigenic or antitumorigenic effects. The roles and mechanisms of PPARg activation in esophageal cancer remain unclarified. EC109 and TE10 esophageal cancer cells were treated with 0, 10, 20 and 40 mM of PPARg agonist rosiglitazone (RGZ) for 24, 48, and 72 h, and the cell viability and apoptosis were detected using methyl thiazolyl tetrazolium (MTT) assay and Flow cytometric (FCM) analysis, respectively. Moreover, the effects of inhibition of PPARg by antagonist or specific RNA interference on cell viability, apoptosis, the Toll-like receptor 4 (TLR4) and mitogen-activated protein kinase (MAPK) pathways were evaluated. Additionally, the effect of TLR4 signaling on the MAPK pathway, cell viability and apoptosis was assessed. The results showed that RGZ suppressed proliferation and induced apoptosis of esophageal cancer cells, which could be partly restored by inactivation of PPARg. RGZ suppressed the MAPK and TLR4 pathways, and the inhibitory effect could be counteracted by PPARg antagonist or specific RNA interference. We also suggested that MAPK activation was regulated by the TLR4 pathway and that blocking the TLR4 and MAPK pathways significantly suppressed proliferation and induced apoptosis of esophageal cancer cells. In conclusion, our data suggested that activation of PPARg suppressed proliferation and induced apoptosis of esophageal cancer cells by inhibiting TLR4-dependent MAPK pathway.

  16. 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

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

    PubMed

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

    2015-06-22

    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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Isorhamnetin prevent endothelial cell injuries from oxidized LDL via activation of p38MAPK.

    PubMed

    Bao, Meihua; Lou, Yijia

    2006-10-10

    The present investigation was undertaken to determine the protective effects of isorhamnetin on endothelial cell line EA.hy926 injuries induced by oxidized low-density lipoprotein (ox-LDL) and to uncover some of the underlying mechanisms of these effects. Indices such as cell viability, lactate dehydrogenase (LDH), and nitric oxide (NO) release were measured to evaluate the protective effects of isorhamnetin. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, superoxide dismutase (SOD), superoxide and reactive oxygen species (ROS) generation were also detected to evaluate the antioxidant effects of isorhamnetin. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to confirm the expression of endothelial nitric oxide synthase (eNOS) mRNA and lectin-like ox-LDL receptor-1 mRNA. Western blotting was used to evaluate the protein expression of this receptor and eNOS, as well as p38-mitogen-activated protein kinase (p38MAPK) phosphorylation and NF-kappaB p65 translocation. As a result, cell viability decreased significantly (P<0.01) after 24 h treatment with ox-LDL, accompanied with apparent secretion disorders such as NO reduction and LDH increase. Pretreatment with isorhamnetin resulted in remarkable increase of cell viability (P<0.05) and modulation of secretion disorders mediated by ox-LDL in a concentration-dependent manner. Besides, ox-LDL led to upregulation of lectin-like ox-LDL receptor-1, phosphorylation of p38MAPK, translocation of NF-kappaB, and downregulation of the eNOS expression in endothelial cells. Isorhamnetin pretreatment inhibited the ox-LDL-induced downregulation of eNOS, upregulation of lectin-like ox-LDL receptor-1, phosphorylation of the p38MAPK and translocation of NF-kappaB. Moreover, isorhamnetin exhibited strong antioxidant activity, which was shown by its inhibition effects on ox-LDL-induced superoxide, ROS overproduction and significant SOD reduction. The data indicated the protective effects of isorhamnetin on

  4. Sesamin suppresses activation of microglia and p44/42 MAPK pathway, which confers neuroprotection in rat intracerebral hemorrhage.

    PubMed

    Ohnishi, M; Monda, A; Takemoto, R; Matsuoka, Y; Kitamura, C; Ohashi, K; Shibuya, H; Inoue, A

    2013-03-01

    Thrombin plays important roles in the pathology of intracerebral hemorrhage (ICH). The recruitment of activated microglia, accompanied by thrombin-induced phosphorylation of the mitogen-activated protein kinase (MAPK) family, contributes to ICH-associated neuron loss. Here we investigated the possibility that sesamin, a lignan of sesame seed oil, is a natural candidate as an inhibitor of microglial activation and MAPK pathways under ICH insults. Sesamin (30-100 μM) suppressed thrombin-induced nitric oxide (NO) production by primary-cultured rat microglia via inhibition of inducible NO synthase (iNOS) protein expression, independently of the antioxidative effect. Sesamin selectively inhibited p44/42 MAPK phosphorylation in the MAPK family (p38 and p44/42) involved in iNOS protein expression in primary-cultured rat microglia. An in vivo rat ICH model was prepared by intrastriatal injection of 0.20U collagenase type IV unilaterally. ICH evoked the phosphorylation of p44/42 MAPK, microglial proliferation with morphological change into the activated ameboid form, and neuron loss. The phosphorylation of p44/42 MAPK was inhibited by intracerebroventricular administration of 30-nmol sesamin. Sesamin prevented ICH-induced increase of microglial cells in the perihematomal area. Notably, ramified microglia, the resting morphology, were observed in brain sections of the animals administrated sesamin. Sesamin furthermore achieved neuroprotection in the perihematomal area but not in the hematomal center. These results suggest that sesamin is a promising natural product as a novel therapeutic strategy based on the regulation of microglial activities accompanied by the activated p44/42 MAPK pathway in ICH.

  5. 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

    2016-08-24

    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.

  6. Surfactin inducing mitochondria-dependent ROS to activate MAPKs, NF-κB and inflammasomes in macrophages for adjuvant activity.

    PubMed

    Gan, Ping; Gao, Zhenqiu; Zhao, Xiuyun; Qi, Gaofu

    2016-12-14

    Surfactin, a natural lipopeptide, can be used both as parenteral and non-parenteral adjuvant for eliciting immune response. However, the mechanisms that confer its adjuvant properties have not been fully explored. By staining with NHS-Rhodamine B labeled surfactin and Mito-Tracker Green, we found surfactin could penetrate into macrophages to bind with mitochondria, following induce ROS that could be inhibited by mitochondria-dependent ROS inhibitor. ROS enhanced p38 MAPK and JNK expression, as well their phorsphorylation, following activated NF-κB nuclear translocation in macrophages that was obviously inhibited by mitochondria-dependent ROS inhibitor. However, inhibition of ROS production only weakened p38 MAPK and JNK expression, but not their phosphorylation in macrophages. As a result, surfaction could activate NF-κB to release TNF-α by the mitochondria-dependent ROS signalling pathway. ROS also induced macrophages apoptosis to release endogenous danger signals, following activated inflammasomes of NLRP1, NLRP3, IPAF and AIM2 in vitro and only NLRP1 in vivo, as well caspase-1 and IL-1 in macrophages, which were significantly inhibited by pre-treatment with ROS inhibitors. Collectively, surfactin as a kind of non-pathogen-associated molecular patterns, modulates host innate immunity by multiple signalling pathways, including induction of mitochondria-dependent ROS, activating MAPKs and NF-κB, and inducing cell apoptosis to realease endogenous danger signals for activation of inflammasomes.

  7. Surfactin inducing mitochondria-dependent ROS to activate MAPKs, NF-κB and inflammasomes in macrophages for adjuvant activity

    PubMed Central

    Gan, Ping; Gao, Zhenqiu; Zhao, Xiuyun; Qi, Gaofu

    2016-01-01

    Surfactin, a natural lipopeptide, can be used both as parenteral and non-parenteral adjuvant for eliciting immune response. However, the mechanisms that confer its adjuvant properties have not been fully explored. By staining with NHS-Rhodamine B labeled surfactin and Mito-Tracker Green, we found surfactin could penetrate into macrophages to bind with mitochondria, following induce ROS that could be inhibited by mitochondria-dependent ROS inhibitor. ROS enhanced p38 MAPK and JNK expression, as well their phorsphorylation, following activated NF-κB nuclear translocation in macrophages that was obviously inhibited by mitochondria-dependent ROS inhibitor. However, inhibition of ROS production only weakened p38 MAPK and JNK expression, but not their phosphorylation in macrophages. As a result, surfaction could activate NF-κB to release TNF-α by the mitochondria-dependent ROS signalling pathway. ROS also induced macrophages apoptosis to release endogenous danger signals, following activated inflammasomes of NLRP1, NLRP3, IPAF and AIM2 in vitro and only NLRP1 in vivo, as well caspase-1 and IL-1 in macrophages, which were significantly inhibited by pre-treatment with ROS inhibitors. Collectively, surfactin as a kind of non-pathogen-associated molecular patterns, modulates host innate immunity by multiple signalling pathways, including induction of mitochondria-dependent ROS, activating MAPKs and NF-κB, and inducing cell apoptosis to realease endogenous danger signals for activation of inflammasomes. PMID:27966632

  8. Molecular basis of MAPK-activated protein kinase 2:p38 assembly

    PubMed Central

    White, Andre; Pargellis, Christopher A.; Studts, Joey M.; Werneburg, Brian G.; Farmer, Bennett T.

    2007-01-01

    p38 MAPK and MAPK-activated protein kinase 2 (MK2) are key components of signaling pathways leading to many cellular responses, notably the proinflammatory cytokine production. The physical association of p38α isoform and MK2 is believed to be physiologically important for this signaling. We report the 2.7-Å resolution crystal structure of the unphosphorylated complex between p38α and MK2. These protein kinases bind “head-to-head,” present their respective active sites on approximately the same side of the heterodimer, and form extensive intermolecular interactions. Among these interactions, the MK2 Ile-366–Ala-390, which includes the bipartite nuclear localization signal, binds to the p38α-docking region. This binding supports the involvement of noncatalytic regions to the tight binding of the MK2:p38α binary assembly. The MK2 residues 345–365, containing the nuclear export signal, block access to the p38α active site. Some regulatory phosphorylation regions of both protein kinases engage in multiple interactions with one another in this complex. This structure gives new insights into the regulation of the protein kinases p38α and MK2, aids in the better understanding of their known cellular and biochemical studies, and provides a basis for understanding other regulatory protein–protein interactions involving signal transduction proteins. PMID:17395714

  9. Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases (MKKs) and mitogen activated kinase phosphatase-1 (MKP-1) in microglial cells

    PubMed Central

    Crittenden, Patrick L.; Filipov, Nikolay M.

    2010-01-01

    Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK, and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e., MKK-3/6, MKK-1/2, and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250μM), LPS (100 ng/ml), or Mn+LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn+LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn+LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn+LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4’s phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn+LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn+LPS markedly increased TNF-α , IL-6, and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6, and MKK-4 are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs farthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect. PMID:20589745

  10. Zinc rescues obesity-induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress-activated BCL10/CARD9/p38 MAPK pathway.

    PubMed

    Wang, Shudong; Gu, Junlian; Xu, Zheng; Zhang, Zhiguo; Bai, Tao; Xu, Jianxiang; Cai, Jun; Barnes, Gregory; Liu, Qiu-Ju; Freedman, Jonathan H; Wang, Yonggang; Liu, Quan; Zheng, Yang; Cai, Lu

    2017-02-03

    Obesity often leads to obesity-related cardiac hypertrophy (ORCH), which is suppressed by zinc-induced inactivation of p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4-week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B-cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate-treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate-induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation.

  11. Solution phase parallel synthesis and evaluation of MAPK inhibitory activities of close structural analogues of a Ras pathway modulator.

    PubMed

    Lu, Yingchun; Sakamuri, Sukumar; Chen, Quin-Zene; Keng, Yen-Fang; Khazak, Vladimir; Illgen, Katrin; Schabbert, Silke; Weber, Lutz; Menon, Sanjay R

    2004-08-02

    A solution phase parallel synthesis approach was undertaken to rapidly explore the structure-activity relationship of an inhibitor of the Ras/Raf protein interaction identified from a small molecule compound library. Evaluation of the MAPK pathway signaling inhibitory activity of the synthesized analogues as well as their antiproliferative activity and ability to inhibit soft agar growth were performed.

  12. MALT1 Inhibition of Oral Carcinoma Cell Invasion and ERK/MAPK Activation.

    PubMed

    Chiba, T; Soeno, Y; Shirako, Y; Sudo, H; Yagishita, H; Taya, Y; Kawashiri, S; Okada, Y; Imai, K

    2016-04-01

    The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)-κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)-expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression.

  13. The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines.

    PubMed

    Charlaftis, Nikolaos; Suddason, Tesha; Wu, Xuefeng; Anwar, Saba; Karin, Michael; Gallagher, Ewen

    2014-11-03

    Unlike the other MAP3Ks, MEKK1 (encoded by Map3k1) contains a PHD motif. To understand the role of this motif, we have created a knockin mutant of mouse Map3k1 (Map3k1(m) (PHD)) with an inactive PHD motif. Map3k1(m) (PHD) ES cells demonstrate that the MEKK1 PHD controls p38 and JNK activation during TGF-β, EGF and microtubule disruption signalling, but does not affect MAPK responses to hyperosmotic stress. Protein microarray profiling identified the adaptor TAB1 as a PHD substrate, and TGF-β- or EGF-stimulated Map3k1(m) (PHD) ES cells exhibit defective non-canonical ubiquitination of MEKK1 and TAB1. The MEKK1 PHD binds and mediates the transfer of Lys63-linked poly-Ub, using the conjugating enzyme UBE2N, onto TAB1 to regulate TAK1 and MAPK activation by TGF-β and EGF. Both the MEKK1 PHD and TAB1 are critical for ES-cell differentiation and tumourigenesis. Map3k1(m) (PHD) (/+) mice exhibit aberrant cardiac tissue, B-cell development, testis and T-cell signalling.

  14. 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.

  15. 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

  16. B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis

    PubMed Central

    Chen, Xuqin; Li, Yan; Blankson, Siobhan; Liu, Min; Huang, Danping; Redmond, H. Paul; Huang, Jing; Wang, Jiang Huai; Wang, Jian

    2017-01-01

    The costimulatory protein B7-H3 has been shown to play a contributory role in the development and progression of experimental pneumococcal meningitis by augmentation of the innate immunity-associated inflammatory response via a TLR2-dependent manner. This study aimed to clarify the component(s) of TLR2-mediated signal transduction pathways responsible for B7-H3-augmented inflammatory response and subsequent brain damage during experimental pneumococcal meningitis. Administration of B7-H3 did not augment expression of TLR2 and other TLR2 upstream components, but led to an enhanced formation of MyD88-IRAK immunocomplex in the brain of S. pneumoniae-infected mice. Furthermore, B7-H3 substantially augmented S. pneumoniae-induced activation of TLR2 downstream NF-κB p65 and MAPK p38 pathways in the brain of S. pneumoniae-infected mice. Notably, blockage of NF-κB p65 and/or MAPK p38 with their specific inhibitors strongly attenuated B7-H3-amplified inflammatory response with significantly reduced proinflammatory cytokine and chemokine production, and markedly ameliorated B7-H3-exacerbated disruption of blood-brain barrier and severity of disease status in S. pneumoniae-infected mice. These results indicate that targeting NF-κB p65 and/or MAPK p38 may represent a promising therapeutic option for amelioration of overwhelming inflammatory response-associated brain injury frequently observed during pneumococcal meningitis. PMID:28141831

  17. p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic β-Cells

    PubMed Central

    Šrámek, Jan; Němcová-Fürstová, Vlasta; Balušíková, Kamila; Daniel, Petr; Jelínek, Michael; James, Roger F.; Kovář, Jan

    2016-01-01

    Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic β-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis. PMID:26861294

  18. 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.

  19. RAS/MAPK Activation Drives Resistance to Smo Inhibition, Metastasis, and Tumor Evolution in Shh Pathway-Dependent Tumors.

    PubMed

    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

    2015-09-01

    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 the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.

  20. 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

  1. MAPK/AP-1-Targeted Anti-Inflammatory Activities of Xanthium strumarium.

    PubMed

    Hossen, Muhammad Jahangir; Kim, Mi-Yeon; Cho, Jae Youl

    2016-01-01

    Xanthium strumarium L. (Asteraceae), a traditional Chinese medicine, is prescribed to treat arthritis, bronchitis, and rhinitis. Although the plant has been used for many years, the mechanism by which it ameliorates various inflammatory diseases is not yet fully understood. To explore the anti-inflammatory mechanism of methanol extracts of X. strumarium (Xs-ME) and its therapeutic potential, we used lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells and human monocyte-like U937 cells as well as a LPS/D-galactosamine (GalN)-induced acute hepatitis mouse model. To find the target inflammatory pathway, we used holistic immunoblotting analysis, reporter gene assays, and mRNA analysis. Xs-ME significantly suppressed the up-regulation of both the activator protein (AP)-1-mediated luciferase activity and the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1[Formula: see text], IL-6, and tumor necrosis factor (TNF)-[Formula: see text]. Moreover, Xs-ME strongly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW264.7 and U937 cells. Additionally, these results highlighted the hepatoprotective and curative effects of Xs-ME in a mouse model of LPS/D-GalN-induced acute liver injury, as assessed by elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and histological damage. Therefore, our results strongly suggest that the ethnopharmacological roles of Xs-ME in hepatitis and other inflammatory diseases might result from its inhibitory activities on the inflammatory signaling of MAPK and AP-1.

  2. 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

  3. Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway

    PubMed Central

    Zhang, Tao; Wang, Zhenlei; Qin, Xuan; Jing, Xiaoqian; Wu, Haoxuan; Ji, Xiaopin; He, Yonggang; Zhao, Ren

    2017-01-01

    Cortactin (CTTN) is overexpressed in various tumors, including head and neck squamous cell carcinoma and colorectal cancer (CRC), and can serve as a biomarker of cancer metastasis. We observed that CTTN promotes cancer cell proliferation in vitro and increases CRC tumor xenograft growth in vivo. CTTN expression increases EGFR protein levels and enhances the activation of the MAPK signaling pathway. CTTN expression also inhibits the ubiquitin-mediated degradation of EGFR by suppressing the coupling of c-Cbl with EGFR. CoIP experiments indicate CTTN can interact with c-Cbl in CRC cells. These results demonstrate that CTTN promotes the proliferation of CRC cells and suppresses the degradation of EGFR. PMID:27903975

  4. Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway.

    PubMed

    Sha, Xiaojin; Meng, Shu; Li, Xinyuan; Xi, Hang; Maddaloni, Massimo; Pascual, David W; Shan, Huimin; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-feng

    2015-07-31

    Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.

  5. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer

    PubMed Central

    Carracedo, Arkaitz; Ma, Li; Teruya-Feldstein, Julie; Rojo, Federico; Salmena, Leonardo; Alimonti, Andrea; Egia, Ainara; Sasaki, Atsuo T.; Thomas, George; Kozma, Sara C.; Papa, Antonella; Nardella, Caterina; Cantley, Lewis C.; Baselga, Jose; Pandolfi, Pier Paolo

    2008-01-01

    Numerous studies have established a causal link between aberrant mammalian target of rapamycin (mTOR) activation and tumorigenesis, indicating that mTOR inhibition may have therapeutic potential. In this study, we show that rapamycin and its analogs activate the MAPK pathway in human cancer, in what represents a novel mTORC1-MAPK feedback loop. We found that tumor samples from patients with biopsy-accessible solid tumors of advanced disease treated with RAD001, a rapamycin derivative, showed an administration schedule–dependent increase in activation of the MAPK pathway. RAD001 treatment also led to MAPK activation in a mouse model of prostate cancer. We further show that rapamycin-induced MAPK activation occurs in both normal cells and cancer cells lines and that this feedback loop depends on an S6K-PI3K-Ras pathway. Significantly, pharmacological inhibition of the MAPK pathway enhanced the antitumoral effect of mTORC1 inhibition by rapamycin in cancer cells in vitro and in a xenograft mouse model. Taken together, our findings identify MAPK activation as a consequence of mTORC1 inhibition and underscore the potential of a combined therapeutic approach with mTORC1 and MAPK inhibitors, currently employed as single agents in the clinic, for the treatment of human cancers. PMID:18725988

  6. 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...

  7. 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

  8. 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

  9. Adiporedoxin suppresses endothelial activation via inhibiting MAPK and NF-κB signaling

    PubMed Central

    He, Hui; Guo, Fang; Li, Yong; Saaoud, Fatma; Kimmis, Brooks D.; Sandhu, Jeena; Fan, Michelle; Maulik, Dev; Lessner, Susan; Papasian, Christopher J.; Fan, Daping; Jiang, Zhisheng; Fu, Mingui

    2016-01-01

    Adiporedoxin (Adrx) is a recently discovered redox regulatory protein that is preferentially expressed in adipose tissue and plays a critical role in the regulation of metabolism via its modulation of adipocyte protein secretion. We here report that Adrx suppresses endothelial cell activation via inhibiting MAPK and NF-kB signaling pathways. Adrx is constitutively expressed in human vascular endothelial cells, and significantly induced by a variety of stimuli such as TNFα, IL-1β, H2O2 and OxLDL. Overexpression of Adrx significantly attenuated TNFα-induced expression of VCAM-1 and ICAM-1, and thus reduced monocyte adherence to human umbilical vein endothelial cells (HUVECs). Conversely, siRNA-mediated knockdown of Adrx increased TNFα-induced expression of adhesion molecules and monocyte adherence to HUVECs. Furthermore, forced expression of Adrx decreased TNFα-induced activation of ERK1/2, JNK, p38 and IKKs in HUVECs. Adrx mutant in the CXXC motif that lost its anti-redox activity is less efficient than the wild-type Adrx, suggesting that Adrx-mediated inhibition of endothelial activation is partially dependent on its antioxidant activity. Finally, Adrx expression was markedly increased in human atheroma compared with normal tissue from the same carotid arteries. These results suggest that Adrx is an endogenous inhibitor of endothelial activation, and might be a therapeutic target for vascular inflammatory diseases. PMID:27941911

  10. Daily sesame oil supplementation attenuates local renin-angiotensin system via inhibiting MAPK activation and oxidative stress in cardiac hypertrophy.

    PubMed

    Liu, Chuan-Teng; Liu, Ming-Yie

    2017-04-01

    The renin-angiotensin system (RAS) is involved in the development of left ventricular hypertrophy (LVH) by which increases cardiac morbidity and mortality. Activation of mitogen-activated protein kinases (MAPKs) and oxidative stress are important in RAS-mediated cardiac hypertrophy. Sesame oil, a potent antioxidant, attenuates hypertension-dependent LVH. We examined the protective role of sesame oil on RAS-mediated MAPK activation and oxidative stress in rats. We induced LVH using a hypertensive model by subcutaneously injecting deoxycorticosterone acetate (DOCA; 15 mg/ml/kg in mineral oil; twice weekly for 5 weeks) and supplementing with 1% sodium chloride drinking water (DOCA/salt) to uninephrectomized rats. Sesame oil was gavaged (0.5 or 1 ml/kg/day for 7 days) after 4 weeks of DOCA/salt treatment. Cardiac histopathology, RAS parameters, expression of MAPKs, reactive oxygen species and lipid peroxidation were assessed 24 h after the last dose of sesame oil. Sesame oil significantly decreased the size of cardiomyocytes and the levels of cardiac renin, angiotensin-converting enzyme and angiotensin II. In addition, sesame oil down-regulated the expression of angiotensin type 1 receptor, JNK and p38 MAPK and apoptosis signal regulating kinase 1, c-Fos and c-Jun in rats receiving DOCA/salt. Furthermore, the induction of nicotinamide adenine dinucleotide phosphate oxidase, superoxide anion and hydroxyl radical and lipid peroxidation by DOCA/salt were inhibited by sesame oil. Sesame oil modulates cardiac RAS to ameliorate LVH by inhibiting MAPK activation and lowering oxidative stress.

  11. Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection

    PubMed Central

    Klemm, Carolin; Bruchhagen, Christin; van Krüchten, Andre; Niemann, Silke; Löffler, Bettina; Peters, Georg; Ludwig, Stephan; Ehrhardt, Christina

    2017-01-01

    Bacterial super-infections are a major complication of influenza virus (IV) infections and often lead to severe pneumonia. One hallmark of IV-associated Staphylococcus aureus (S. aureus) infection is rapid progression to a serious disease outcome. Changes in immune and inflammatory host responses increase morbidity and complicate efficient therapy. A key player during inflammation is the multifunctional cytokine IL-6. Although increased IL-6 levels have been observed after severe disease upon IV and/or bacterial super-infection, the underlying molecular mechanisms still remain to be elucidated. In the present study, we focused on cellular signalling pathways regulating IL-6 production upon IV/S. aureus super-infection. Additionally, infection with viable bacteria was mimicked by lipoteichoic acid stimulation in this model. Analyses of cellular signalling mechanisms revealed synergistically increased activation of the MAPK p38 as well as enhanced phosphorylation of the MAPKs ERK1/2 and JNK in the presence of super-infecting bacteria. Interestingly, inhibition of MAPK activity indicated a strong dependence of IL-6 expression on p38 and ERK1/2, while the MAPK JNK seems not to be involved. Thus, our results provide new molecular insights into the regulation of IL-6, a marker of severe disease, which might contribute to the lethal synergism of IV and S. aureus. PMID:28195157

  12. Low cell cholesterol levels increase NFkappaB activity through a p38 MAPK-dependent mechanism.

    PubMed

    Calleros, Laura; Lasa, Marina; Toro, María J; Chiloeches, Antonio

    2006-12-01

    Cholesterol, p38 MAPK and NFkappaB have been shown to participate in inflammation and cellular differentiation. Here, we examined the effect of cholesterol on NFkappaB-dependent transcription and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in NFkappaB-dependent transcription, NFkappaB-DNA binding, IkappaBalpha degradation and p65/NFkappaB translocation to the nucleus, and the addition of exogenous cholesterol reversed these effects. Previously, we have shown that low cell cholesterol levels activate p38 MAPK. Here, we found that inhibition of p38 MAPK with the specific inhibitor SB203580 blocked the increase in NFkappaB activity, IkappaBalpha degradation and p65/NFkappaB translocation to the nucleus induced by cholesterol depletion. Moreover, the inhibition of the p38 MAPK downstream effector MSK1 with the specific inhibitor H89, or the overexpression of a kinase defective MSK1 abrogated the NFkappaB-dependent transcription induced by cholesterol depletion. On the other hand, the transactivation potential of p65/NFkappaB depends on phosphorylation of S276 by MSK1. We observed that cholesterol depletion increased the p65/NFkappaB transactivation capacity. This effect was reversed by cell cholesterol repletion or incubation with the SB203580 inhibitor. Moreover, the expression of a p65/NFkappaB S276A mutant was insensitive to cholesterol depletion. Together, our results demonstrate that cholesterol depletion induces NFkappaB transcriptional activity, not only by affecting the IkappaBalpha degradation and the translocation of p65/NFkappaB to the nucleus, but also regulating the p65/NFkappaB transactivating potential through a p38 MAPK/MSK1 mediated pathway.

  13. MAPK-Activated Protein Kinase 2 Contributes to Clostridium difficile-Associated Inflammation

    PubMed Central

    Bobo, Linda D.; El Feghaly, Rana E.; Chen, Yee-Shiuan; Dubberke, Erik R.; Han, Zhuolin; Baker, Alexandra H.; Li, Jinmei; Burnham, Carey-Ann D.

    2013-01-01

    Clostridium difficile infection (CDI) results in toxin-induced epithelial injury and marked intestinal inflammation. Fecal markers of intestinal inflammation correlate with CDI disease severity, but regulation of the inflammatory response is poorly understood. Previous studies demonstrated that C. difficile toxin TcdA activates p38 kinase in tissue culture cells and mouse ilium, resulting in interleukin-8 (IL-8) release. Here, we investigated the role of phosphorylated mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2 kinase, pMK2), a key mediator of p38-dependent inflammation, in CDI. Exposure of cultured intestinal epithelial cells to the C. difficile toxins TcdA and TcdB resulted in p38-dependent MK2 activation. Toxin-induced IL-8 and GROα release required MK2 activity. We found that p38 and MK2 are activated in response to other actin-disrupting agents, suggesting that toxin-induced cytoskeleton disruption is the trigger for kinase-dependent cytokine response. Phosphorylated MK2 was detected in the intestines of C. difficile-infected hamsters and mice, demonstrating for the first time that the pathway is activated in infected animals. Furthermore, we found that elevated pMK2 correlated with the presence of toxigenic C. difficile among 100 patient stool samples submitted for C. difficile testing. In conclusion, we find that MK2 kinase is activated by TcdA and TcdB and regulates the expression of proinflammatory cytokines. Activation of p38-MK2 in infected animals and humans suggests that this pathway is a key driver of intestinal inflammation in patients with CDI. PMID:23264053

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

    PubMed Central

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

    2015-01-01

    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. PMID:25909220

  15. Baicalin positively regulates osteoclast function by activating MAPK/Mitf signalling.

    PubMed

    Lu, Li; Rao, Li; Jia, Huanhuan; Chen, Jun; Lu, Xingyan; Yang, Guozhu; Li, Qingnan; Lee, Kenneth Ka Ho; Yang, Li

    2017-02-03

    Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose-dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up-regulation of downstream osteoclast-related target genes expression. The baicalin-induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p-ERK (U0126) and the Mitf-specific siRNA, respectively. Protein-ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p-ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p-ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.

  16. 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.

  17. MAPK Scaffold IQGAP1 Binds the EGF Receptor and Modulates Its Activation*

    PubMed Central

    McNulty, Dean E.; Li, Zhigang; White, Colin D.; Sacks, David B.; Annan, Roland S.

    2011-01-01

    Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca2+ attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser1443, a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation. PMID:21349850

  18. 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.

  19. AKT-phosphorylated FOXO1 suppresses ERK activation and chemoresistance by disrupting IQGAP1-MAPK interaction.

    PubMed

    Pan, Chun-Wu; Jin, Xin; Zhao, Yu; Pan, Yunqian; Yang, Jing; Karnes, R Jeffrey; Zhang, Jun; Wang, Liguo; Huang, Haojie

    2017-03-09

    Nuclear FOXO proteins act as tumor suppressors by transcriptionally activating genes involved in apoptosis and cell cycle arrest, and these anticancer functions are inhibited by AKT-induced phosphorylation and cytoplasmic sequestration of FOXOs. We found that, after AKT-mediated phosphorylation at serine 319, FOXO1 binds to IQGAP1, a hub for activation of the MAPK pathway, and impedes IQGAP1-dependent phosphorylation of ERK1/2 (pERK1/2). Conversely, decreased FOXO1 expression increases pERK1/2 in cancer cell lines and correlates with increased pERK1/2 levels in patient specimens and disease progression. Treatment of cancer cells with PI3K inhibitors or taxane causes FOXO1 localization in the nucleus, increased expression of pERK1/2, and drug resistance. These effects are reversed by administering a small FOXO1-derived phospho-mimicking peptide inhibitor in vitro and in mice. Our results show a tumor suppressor role of AKT-phosphorylated FOXO1 in the cytoplasm and suggest that this function of FOXO1 can be harnessed to overcome chemoresistance in cancer.

  20. Hypoxia-Inducible Factor-1alpha and MAPK Co-Regulate Activation of Hepatic Stellate Cells upon Hypoxia Stimulation

    PubMed Central

    Guan, Fei; Xiao, Yan; Deng, Jing; Chen, Huoying; Chen, Xiaolin; Li, Jianrong; Huang, Hanju; Shi, Chunwei

    2013-01-01

    Background Hepatic stellate cell (HSC) plays a key role in pathogenesis of liver fibrosis. During liver injury, hypoxia in local micro-environment is inevitable. Hif-1α is the key transcriptional regulation factor that induces cell’s adaptive responses to hypoxia. Recently, it was reported that MAPK is involved in regulation of Hif-1α activity. Aims To explore whether Hif-1α regulates HSC activation upon hypoxia, and whether MAPK affects Hif-1α-regulated signaling cascades, thus providing new targets for preventing liver fibrosis. Methods Hif-1α expression in livers of Schistosomajaponicum infected BALB/c mice was detected with western blot and immunohistochemistry. A rat cell line of HSC, HSC-T6, was cultured in 1% oxygen. HSC activation, including F-actin reorganization, increase of vimentin and α-SMA, was detected with western blot or immunocytochemistry. Cells were transfected with specific siRNA to Hif-1α, expression of activation markers, transcription of fibrosis-promoting cytokines, secretion of collagen I were detected with western blot, Real Time PCR and ELISA. Lysate from HSC-T6 cells pretreated with PD98059, a specific MEK1 pharmacological inhibitor, was subjected to detect Hif-1α ubiquitination and nuclear translocation with western blot and immunoprecipitation. Results and Conclusions Hif-1α apparently increased in liver tissues of Schistosomajaponicum infected mice. 1% O2 induced F-actin reorganization, increase of Hif-1α, vimentin and α-SMA in HSC-T6 cells. Hif-1α Knockdown inhibited HSC-T6 activation, transcription of IL-6, TGF-β and CTGF and secretion of collagen I from HSC-T6 cells upon hypoxia. Inhibition of MAPK phosphorylation enhanced Hif-1α ubiquitination, and inhibited Hif-1α translocation into nucleus. Conclusively, Hif-1α and MAPK participate in HSC activation upon hypoxia. PMID:24040163

  1. Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association

    PubMed Central

    Conti, Filippo; Boucherit, Nicolas; Baldassarre, Veronica; Trouplin, Virginie; Toman, Rudolf; Mottola, Giovanna; Mege, Jean-Louis; Ghigo, Eric

    2015-01-01

    To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association. PMID:25610812

  2. 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

  3. Inhibition of adipogenic differentiation of bone marrow mesenchymal stem cells by erythropoietin via activating ERK and P38 MAPK.

    PubMed

    Liu, G X; Zhu, J C; Chen, X Y; Zhu, A Z; Liu, C C; Lai, Q; Chen, S T

    2015-06-26

    We examined whether erythropoietin (EPO) can inhibit adipogenic differentiation of mesenchymal stem cells (MSCs) in the mouse bone marrow and its underlying mechanism. We separated and extracted mouse bone marrow MSCs and induced adipogenic differen-tiation using 3-isobutyl-1-methylxanthine, insulin, and dexamethasone. Different concentrations of EPO were added to the cells and observed by Oil Red O staining on the 20th day to quantitatively analyze the degree of cell differentiation. mRNA expression levels of peroxysome proliferator-activated receptor γ (PPARγ), CCAAT enhancer binding protein α, and adiponectin were analyzed by real-time quantitative polymerase chain reaction, and the activity of PPARγ, extracellular sig-nal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) were determined by western blotting. EPO significantly inhibited adipogenic differentiation of MSCs after 20 days and reduced absorbance values by Oil Red O staining without affecting proliferation activity. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponec-tin during adipogenesis and increased protein phosphorylation of ERK, p38 MAPK, and PPARγ during differentiation. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponectin by increasing protein phosphor-ylation of ERK, p38 MAPK, and PPARγ during differentiation, which inhibited adipogenic differentiation of MSCs.

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

    PubMed Central

    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. PMID:26269757

  5. 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.

  6. Discoidin domain receptor 1 promotes Th17 cell migration by activating the RhoA/ROCK/MAPK/ERK signaling pathway

    PubMed Central

    Azreq, Mohammed-Amine El; Kadiri, Maleck; Boisvert, Marc; Pagé, Nathalie; Tessier, Philippe A.; Aoudjit, Fawzi

    2016-01-01

    Effector T cell migration through the tissue extracellular matrix (ECM) is an important step of the adaptive immune response and in the development of inflammatory diseases. However, the mechanisms involved in this process are still poorly understood. In this study, we addressed the role of a collagen receptor, the discoidin domain receptor 1 (DDR1), in the migration of Th17 cells. We showed that the vast majority of human Th17 cells express DDR1 and that silencing DDR1 or using the blocking recombinant receptor DDR1:Fc significantly reduced their motility and invasion in three-dimensional (3D) collagen. DDR1 promoted Th17 migration by activating RhoA/ROCK and MAPK/ERK signaling pathways. Interestingly, the RhoA/ROCK signaling module was required for MAPK/ERK activation. Finally, we showed that DDR1 is important for the recruitment of Th17 cells into the mouse dorsal air pouch containing the chemoattractant CCL20. Collectively, our results indicate that DDR1, via the activation of RhoA/ROCK/MAPK/ERK signaling axis, is a key pathway of effector T cell migration through collagen of perivascular tissues. As such, DDR1 can contribute to the development of Th17-dependent inflammatory diseases. PMID:27391444

  7. 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

  8. 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...

  9. 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

  10. Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling

    PubMed Central

    Olajide, Olumayokun A.; Aderogba, Mutalib A.; Okorji, Uchechukwu P.; Fiebich, Bernd L.

    2012-01-01

    Bridelia ferruginea is commonly used in traditional African medicine (TAM) for treating various inflammatory conditions. Extracts from the plant have been shown to exhibit anti-inflammatory property in a number of in vivo models. In this study the influence of B. ferruginea (BFE) on the production of PGE2, nitrite, and proinflammatory cytokines from LPS-stimulated BV-2 microglia was investigated. The effects of BFE on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions were evaluated in LPS-activated rat primary microglia. The roles of NF-κB and MAPK signalling in the actions of BFE were also investigated. BFE (25–200 μg) inhibited the production of PGE2, nitrite, tumour necrosis factor-α (TNFα), and interleukin-6 (IL-6) as well as COX-2 and iNOS protein expressions in LPS-activated microglial cells. Further studies to elucidate the mechanism of anti-inflammatory action of BFE revealed interference with nuclear translocation of NF-κBp65 through mechanisms involving inhibition of IκB degradation. BFE prevented phosphorylation of p38, but not p42/44 or JNK MAPK. It is suggested that Bridelia ferruginea produces anti-inflammatory action through mechanisms involving p38 MAPK and NF-κB signalling. PMID:23320030

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

    PubMed

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

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. 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 inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. 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 activation of p44/42 by TBT in NK cells.

  12. Polyubiquitination of Transforming Growth Factor β-activated Kinase 1 (TAK1) at Lysine 562 Residue Regulates TLR4-mediated JNK and p38 MAPK Activation

    PubMed Central

    Chen, I-Ting; Hsu, Pang-Hung; Hsu, Wan-Ching; Chen, Nien-Jung; Tseng, Ping-Hui

    2015-01-01

    Toll-like receptor 4 (TLR4) plays an important role in innate immunity by eliciting inflammation. Upon receptor engagement, transforming growth factor β-activated kinase 1 (TAK1) is an essential mediator that transmits a signal from the receptor to downstream effectors, IκB kinase (IKK) and the mitogen-activated protein kinases (MAPKs), which control the production of inflammatory cytokines. However, the association between phosphorylation and ubiquitination of TAK1 is not yet clear. Here, we examined the crosstalk between phosphorylation and polyubiquitination of TAK1 and further investigated the mechanism of distinct activation of MAPKs and IKK. Inhibition of TAK1 phosphorylation enhanced Lys63-linked polyubiquitination of TAK1. Conversely, ubiquitin modification was counteracted by phospho-mimic TAK1 mutant, T(184,187)D. Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation. Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex. Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway. PMID:26189595

  13. B7-H1 antibodies lose antitumor activity due to activation of p38 MAPK that leads to apoptosis of tumor-reactive CD8+ T cells

    PubMed Central

    Liu, Xin; Wu, Xiaosheng; Cao, Siyu; Harrington, Susan M.; Yin, Peng; Mansfield, Aaron S.; Dong, Haidong

    2016-01-01

    B7-H1 (aka PD-L1) blocking antibodies have been used in treatment of human cancers through blocking B7-H1 expressed by tumor cells; however, their impact on B7-H1 expressing tumor-reactive CD8+ T cells is still unknown. Here, we report that tumor-reactive CD8+ T cells expressing B7-H1 are functional effector cells. In contrast to normal B7-H1 blocking antibody, B7-H1 antibodies capable of activating p38 MAPK lose their antitumor activity by deleting B7-H1+ tumor-reactive CD8+ T cells via p38 MAPK pathway. B7-H1 deficiency or engagement with certain antibody results in more activation of p38 MAPK that leads to T cell apoptosis. DNA-PKcs is a new intracellular partner of B7-H1 in the cytoplasm of activated CD8+ T cells. B7-H1 suppresses p38 MAPK activation by sequestering DNA-PKcs in order to preserve T cell survival. Our findings provide a new mechanism of action of B7-H1 in T cells and have clinical implications in cancer immunotherapy when anti-B7-H1 (PD-L1) antibody is applied. PMID:27824138

  14. Adenosine triphosphate prevents serum deprivation-induced apoptosis in human mesenchymal stem cells via activation of the MAPK signaling pathways.

    PubMed

    Berlier, Jessica L; Rigutto, Sabrina; Dalla Valle, Antoine; Lechanteur, Jessica; Soyfoo, Muhammad S; Gangji, Valerie; Rasschaert, Joanne

    2015-01-01

    Human mesenchymal stem cells (hMSC) are multipotent cells derived from various sources including adipose and placental tissues as well as bone marrow. Owing to their regenerative and immunomodulatory properties, their use as a potential therapeutic tool is being extensively tested. However, one of the major hurdles in using cell-based therapy is the use of fetal bovine serum that can trigger immune responses, viral and prion diseases. The development of a culture medium devoid of serum while preserving cell viability is therefore a major challenge. In this study, we demonstrated that adenosine triphosphate (ATP) restrained serum deprivation-induced cell death in hMSC by preventing caspases 3/7 activation and modulating ERK1/2 and p38 MAPK signaling pathways. We also showed that serum deprivation conditions triggered dephosphorylation of the proapoptotic protein Bad leading to cell death. Adjunction of ATP restored the phosphorylation state of Bad. Furthermore, ATP significantly modulated the expression of proapoptopic and antiapoptotic genes, in favor of an antiapoptotic profile expression. Finally, we established that hMSC released a high amount of ATP in the extracellular medium when cultured in a serum-free medium. Collectively, our results demonstrate that ATP favors hMSC viability in serum deprivation conditions. Moreover, they shed light on the cardinal role of the MAPK pathways, ERK1/2 and p38 MAPK, in promoting hMSC survival.

  15. Epinephrine--via activation of p38-MAPK--abolishes the effect of aspirin on platelet deposition to collagen.

    PubMed

    Mustonen, P; van Willigen, G; Lassila, R

    2001-12-15

    The mechanism by which epinephrine enhances experimental thrombosis in the presence of aspirin is poorly understood. In this study, we set to explore, in aspirinised platelet-rich plasma (PRP), the effect of epinephrine (100 nmol/l) on platelet deposition to immobilised collagen and the subsequent involvement of several intracellular pathways. Under these experimental conditions, which allow platelet aggregation on top of the collagen-adherent platelets, epinephrine increased platelet deposition by 55-86%. This enhancement could be specifically prohibited by the alpha(2A)-adrenoceptor antagonist, atipamezole, the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, and the cytosolic phospholipase A(2) (cPLA(2)) inhibitor, mepacrine. The effect of epinephrine coincided with increased phosphorylation of p38MAPK and cPLA(2) and with arachidonic acid (AA) release from platelet membrane. We conclude that epinephrine enhanced platelet deposition on collagen in aspirinised PRP via a mechanism dependent on both free AA in platelet cytosol (released by cPLA(2)) and p38MAPK.

  16. Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes.

    PubMed

    Lee, Hyun-Seo; Kim, Kyeoung-Hwa; Kim, Eun-Young; Lee, Su-Yeon; Ko, Jung-Jae; Lee, Kyung-Ah

    2016-04-01

    Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.

  17. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB.

    PubMed Central

    Deak, M; Clifton, A D; Lucocq, L M; Alessi, D R

    1998-01-01

    We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB. PMID:9687510

  18. Inhibition of aldehyde dehydrogenase 2 activity enhances antimycin-induced rat cardiomyocytes apoptosis through activation of MAPK signaling pathway.

    PubMed

    Zhang, Peng; Xu, Danling; Wang, Shijun; Fu, Han; Wang, Keqiang; Zou, Yunzeng; Sun, Aijun; Ge, Junbo

    2011-12-01

    Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial-specific enzyme, has been proved to be involved in oxidative stress-induced cell apoptosis, while little is known in cardiomyocytes. This study was aimed at investigating the role of ALDH2 in antimycin A-induced cardiomyocytes apoptosis by suppressing ALDH2 activity with a specific ALDH2 inhibitor Daidzin. Antimycin A (40μg/ml) was used to induce neonatal cardiomyocytes apoptosis. Daidzin (60μM) effectively inhibited ALDH2 activity by 50% without own effect on cell apoptosis, and significantly enhanced antimycin A-induced cardiomyocytes apoptosis from 33.5±4.4 to 56.5±6.4% (Hochest method, p<0.05), and from 57.9±1.9 to 74.0±11.9% (FACS, p<0.05). Phosphorylation of activated MAPK signaling pathway, including extracellular signal-regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38 was also increased in antimycin A and daidzin treated cardiomyocytes compared to the cells treated with antimycin A alone. These findings indicated that modifying mitochondrial ALDH2 activity/expression might be a potential therapeutic option on reducing oxidative insults induced cardiomyocytes apoptosis.

  19. Coordination of Satellite Cell Activation and Self-Renewal by Par-Complex-Dependent Asymmetric Activation of p38α/β MAPK

    PubMed Central

    Troy, Andrew; Cadwallader, Adam B.; Fedorov, Yuri; Tyner, Kristina; Tanaka, Kathleen Kelly; Olwin, Bradley B.

    2014-01-01

    SUMMARY In response to muscle injury, satellite cells activate the p38α/β MAPK pathway to exit quiescence, then proliferate, repair skeletal muscle, and self-renew, replenishing the quiescent satellite cell pool. Although satellite cells are capable of asymmetric division, the mechanisms regulating satellite cell self-renewal are not understood. We found that satellite cells, once activated, enter the cell cycle and a subset undergoes asymmetric division, renewing the satellite cell pool. Asymmetric localization of the Par complex activates p38α/β MAPK in only one daughter cell, inducing MyoD, which permits cell cycle entry and generates a proliferating myoblast. The absence of p38α/β MAPK signaling in the other daughter cell prevents MyoD induction, renewing the quiescent satellite cell. Thus, satellite cells employ a mechanism to generate distinct daughter cells, coupling the Par complex and p38α/β MAPK signaling to link the response to muscle injury with satellite cell self-renewal. PMID:23040480

  20. Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

    PubMed Central

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

    2013-01-01

    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. PMID:23221999

  1. 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

  2. BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana.

    PubMed

    Lee, Min-Chul; Puthumana, Jayesh; Lee, Seung-Hwi; Kang, Hye-Min; Park, Jun Chul; Jeong, Chang-Bum; Han, Jeonghoon; Hwang, Dae-Sik; Seo, Jung Soo; Park, Heum Gi; Om, Ae-Son; Lee, Jae-Seong

    2016-12-01

    Brominated flame retardant, 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana. Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress (e.g. reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR, SREBP, ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene (e.g. ELO3, Δ5-DES, Δ9-DES) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain.

  3. Adverse effects of MWCNTs on life parameters, antioxidant systems, and activation of MAPK signaling pathways in the copepod Paracyclopina nana.

    PubMed

    Kim, Duck-Hyun; Puthumana, Jayesh; Kang, Hye-Min; Lee, Min-Chul; Jeong, Chang-Bum; Han, Jeonghoon; Hwang, Dae-Sik; Kim, Il-Chan; Lee, Jin Wuk; Lee, Jae-Seong

    2016-10-01

    Engineered multi-walled carbon nanotubes (MWCNTs) have received widespread applications in a broad variety of commercial products due to low production cost. Despite their significant commercial applications, CNTs are being discharged to aquatic ecosystem, leading a threat to aquatic life. Thus, we investigated the adverse effect of CNTs on the marine copepod Paracyclopina nana. Additional to the study on the uptake of CNTs and acute toxicity, adverse effects on life parameters (e.g. growth, fecundity, and size) were analyzed in response to various concentrations of CNTs. Also, as a measurement of cellular damage, oxidative stress-related markers were examined in a time-dependent manner. Moreover, activation of redox-sensitive mitogen-activated protein kinase (MAPK) signaling pathways along with the phosphorylation pattern of extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinases (JNK) were analyzed to obtain a better understanding of molecular mechanism of oxidative stress-induced toxicity in the copepod P. nana. As a result, significant inhibition on life parameters and evoked antioxidant systems were observed without ROS induction. In addition, CNTs activated MAPK signaling pathway via ERK, suggesting that phosphorylated ERK (p-ERK)-mediated adverse effects are the primary cause of in vitro and in vivo endpoints in response to CNTs exposure. Moreover, ROS-independent activation of MAPK signaling pathway was observed. These findings will provide a better understanding of the mode of action of CNTs on the copepod P. nana at cellular and molecular level and insight on possible ecotoxicological implications in the marine environment.

  4. 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.

  5. KSR is a scaffold required for activation of the ERK/MAPK module

    PubMed Central

    Roy, François; Laberge, Gino; Douziech, Mélanie; Ferland-McCollough, David; Therrien, Marc

    2002-01-01

    Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. Here, we show that KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, we found that KSR facilitates the phosphorylation of MEK by RAF. We further show that KSR associates independently with RAF and MEK, and that these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair. PMID:11850406

  6. p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability

    PubMed Central

    Fabris, Linda; Berton, Stefania; Pellizzari, Ilenia; Segatto, Ilenia; D’Andrea, Sara; Armenia, Joshua; Bomben, Riccardo; Schiappacassi, Monica; Gattei, Valter; Philips, Mark R.; Vecchione, Andrea; Belletti, Barbara; Baldassarre, Gustavo

    2015-01-01

    The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27kip1-null mice is reverted by concomitant deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting that a CDK-independent function of p27kip1 contributes to the control of cell proliferation. Whether the regulation of MT stability by p27kip1 impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27kip1-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27kip1, by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27kip1 and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway. PMID:26512117

  7. p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability.

    PubMed

    Fabris, Linda; Berton, Stefania; Pellizzari, Ilenia; Segatto, Ilenia; D'Andrea, Sara; Armenia, Joshua; Bomben, Riccardo; Schiappacassi, Monica; Gattei, Valter; Philips, Mark R; Vecchione, Andrea; Belletti, Barbara; Baldassarre, Gustavo

    2015-11-10

    The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

  8. 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.

  9. Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Gerwins, P; Johnson, G L; Henson, P M

    1997-04-01

    Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.

  10. TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

    PubMed

    Yi, Ling; Chandrasekaran, Prabha; Venkatesan, Sundararajan

    2012-01-01

    Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ~0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.

  11. 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.

  12. GITRL modulates the activities of p38 MAPK and STAT3 to promote Th17 cell differentiation in autoimmune arthritis.

    PubMed

    Tang, Xinyi; Tian, Jie; Ma, Jie; Wang, Jiemin; Qi, Chen; Rui, Ke; Wang, Yungang; Xu, Huaxi; Lu, Liwei; Wang, Shengjun

    2016-02-23

    The glucocorticoid-induced TNFR family-related protein (GITR) and its ligand play a critical role in the pathogenesis of autoimmune arthritis by enhancing the Th17 cell response, but their molecular mechanisms remain largely unclear. This study aims to define the role of p38 mitogen-activated protein kinases (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling in GITRL-induced Th17 cells in autoimmune arthritis. We found that the p38 phosphorylation was enhanced by GITRL in activated CD4+T cells, and the p38 inhibitor restrained the GITRL-induced Th17 cell expansion in a dose-dependent manner. Moreover, there was decreased STAT3 activity on Tyr705 and Ser727 with the p38 inhibitor in vitro. Notably, the p38 inhibitor could prevent GITRL-treated arthritis progression and markedly decrease the Th17 cell percentages. The phosphorylation of the Tyr705 site was significantly lower in the GITRL-treated CIA mice administrated with the p38 inhibitor. A significantly higher phosphorylation of p38 was detected in RA patients and had a positive relationship with the serum level of anti-cyclic citrullinated peptide (anti-CCP) antibody. Our findings have indicated that GITRL could promote Th17 cell differentiation by p38 MAPK and STAT3 signaling in autoimmune arthritis.

  13. Xanthohumol induces apoptosis in human malignant glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways.

    PubMed

    Festa, Michela; Capasso, Anna; D'Acunto, Cosimo W; Masullo, Milena; Rossi, Adriano G; Pizza, Cosimo; Piacente, Sonia

    2011-12-27

    The effect of the biologically active prenylated chalcone and potential anticancer agent xanthohumol (1) has been investigated on apoptosis of the T98G human malignant glioblastoma cell line. Compound 1 decreased the viability of T98G cells by induction of apoptosis in a time- and concentration-dependent manner. Apoptosis induced by 1 was associated with activation of caspase-3, caspase-9, and PARP cleavage and was mediated by the mitochondrial pathway, as exemplified by mitochondrial depolarization, cytochrome c release, and downregulation of the antiapoptotic Bcl-2 protein. Xanthohumol induced intracellular reactive oxygen species (ROS), an effect that was reduced by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC). Intracellular ROS production appeared essential for the activation of the mitochondrial pathway and induction of apoptosis after exposure to 1. Oxidative stress due to treatment with 1 was associated with MAPK activation, as determined by ERK1/2 and p38 phosphorylation. Phosphorylation of ERK1/2 and p38 was attenuated using NAC to inhibit ROS production. After treatment with 1, ROS provided a specific environment that resulted in MAPK-induced cell death, with this effect reduced by the ERK1/2 specific inhibitor PD98059 and partially inhibited by the p38 inhibitor SB203580. These findings suggest that xanthohumol (1) is a potential chemotherapeutic agent for the treatment of glioblastoma multiforme.

  14. Ceftiofur impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-{kappa}B and MAPK

    SciTech Connect

    Ci Xinxin; Song Yu; Zeng Fanqin; Zhang Xuemei; Li Hongyu; Wang Xinrui; Cui Junqing Deng Xuming

    2008-07-18

    Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-1{beta} (IL-1{beta}), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10 mg/L of ceftiofur 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-{kappa}B (NF-{kappa}B) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH{sub 2}-terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-{kappa}B translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-{kappa}B and MAPKs signaling in RAW264.7 cells.

  15. Strength of ERK1/2 MAPK Activation Determines Its Effect on Myelin and Axonal Integrity in the Adult CNS

    PubMed Central

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

    2016-01-01

    Myelin growth is a tightly regulated process driven by multiple signals. ERK1/2-MAPK signaling is an important regulator of myelin thickness. Because, in demyelinating diseases, the myelin formed during remyelination fails to achieve normal thickness, increasing ERK1/2 activity in oligodendrocytes is of obvious therapeutic potential for promoting efficient remyelination. However, other studies have suggested that increased levels of ERK1/2 activity could, in fact, have detrimental effects on myelinating cells. Because the strength, duration, or timing of ERK1/2 activation may alter the biological outcomes of cellular responses markedly, here, we investigated the effect of modulating ERK1/2 activity in myelinating cells using transgenic mouse lines in which ERK1/2 activation was upregulated conditionally in a graded manner. We found enhanced myelin gene expression and myelin growth in the adult CNS at both moderate and hyperactivated levels of ERK1/2 when upregulation commenced during developmental myelination or was induced later during adulthood in quiescent preexisting oligodendrocytes, after active myelination is largely terminated. However, a late onset of demyelination and axonal degeneration occurred at hyperelevated, but not moderately elevated, levels regardless of the timing of the upregulation. Similarly, myelin and axonal pathology occurred with elevated ERK1/2 activity in Schwann cells. We conclude that a fine tuning of ERK1/2 signaling strength is critically important for normal oligodendrocyte and Schwann cell function and that disturbance of this balance has negative consequences for myelin and axonal integrity in the long term. Therefore, therapeutic modulation of ERK1/2 activity in demyelinating disease or peripheral neuropathies must be approached with caution. SIGNIFICANCE STATEMENT ERK1/2-MAPK activation in oligodendrocytes and Schwann cells is an important signal for promoting myelin growth during developmental myelination. Here, we show that

  16. MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells.

    PubMed

    Collins, Carol M; Ellis, Joseph A; Holaska, James M

    2017-04-01

    Mutations in the gene encoding emerin cause Emery-Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo.

  17. CAGE, a novel cancer/testis antigen gene, promotes cell motility by activation ERK and p38 MAPK and downregulating ROS.

    PubMed

    Shim, Hyeeun; Shim, Eunsook; Lee, Hansoo; Hahn, Janghee; Kang, Dongmin; Lee, Yun-Sil; Jeoung, Dooil

    2006-06-30

    We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.

  18. Palmitoleic acid prevents palmitic acid-induced macrophage activation and consequent p38 MAPK-mediated skeletal muscle insulin resistance.

    PubMed

    Talbot, Nicola A; Wheeler-Jones, Caroline P; Cleasby, Mark E

    2014-08-05

    Obesity and saturated fatty acid (SFA) treatment are both associated with skeletal muscle insulin resistance (IR) and increased macrophage infiltration. However, the relative effects of SFA and unsaturated fatty acid (UFA)-activated macrophages on muscle are unknown. Here, macrophages were treated with palmitic acid, palmitoleic acid or both and the effects of the conditioned medium (CM) on C2C12 myotubes investigated. CM from palmitic acid-treated J774s (palm-mac-CM) impaired insulin signalling and insulin-stimulated glycogen synthesis, reduced Inhibitor κBα and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in myotubes. p38 MAPK inhibition or siRNA partially ameliorated these defects, as did addition of tumour necrosis factor-α blocking antibody to the CM. Macrophages incubated with both FAs generated CM that did not induce IR, while palmitoleic acid-mac-CM alone was insulin sensitising. Thus UFAs may improve muscle insulin sensitivity and counteract SFA-mediated IR through an effect on macrophage activation.

  19. CCN1 promotes IL-1β production in keratinocytes by activating p38 MAPK signaling in psoriasis

    PubMed Central

    Sun, Yue; Zhang, Jie; Zhai, Tianhang; Li, Huidan; Li, Haichuan; Huo, Rongfen; Shen, Baihua; Wang, Beiqing; Chen, Xiangdong; Li, Ningli; Teng, Jialin

    2017-01-01

    CCN1, an extracellular protein also known as cysteine-rich protein 61 (Cyr61), is a novel pro-inflammatory factor involved in the pathogenesis of rheumatoid arthritis. As an inflammatory disease, psoriasis is characterized by keratinocyte activation-induced epidermal hyperplasia and cytokine-mediated inflammation. We demonstrated in our previous study that CCN1 promoted keratinocyte activation in psoriasis. However, the role of CCN1 in regulating inflammation in psoriasis is still unknown. Here, we showed that CCN1 increased inflammatory cytokine IL-1β production in keratinocytes. Furthermore, endogenous ATP and caspase-1 were required for mature IL-1β production stimulated by CCN1 in keratinocytes. After binding to the receptor of integrin α6β1, CCN1 activated the downstream p38 MAPK signaling pathway, thus inducing the expression of IL-1β. In addition, we inhibited CCN1 function in mouse models of psoriasis, and decreased IL-1β production was observed in vivo. Overall, we showed that CCN1 increased IL-1β production via p38 MAPK signaling, indicating a role for CCN1 protein in regulating inflammation in psoriasis. PMID:28266627

  20. 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.

  1. Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer.

    PubMed

    Okimoto, Ross A; Lin, Luping; Olivas, Victor; Chan, Elton; Markegard, Evan; Rymar, Andrey; Neel, Dana; Chen, Xiao; Hemmati, Golzar; Bollag, Gideon; Bivona, Trever G

    2016-11-22

    Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAF(V600E)-mutant allele, the spectrum of BRAF mutations in LA includes BRAF(V600E) (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAF(G469A) and BRAF(G466V) The presence of BRAF(V600E) in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAF(V600E)-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAF(V600E) and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAF(V600E) that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.

  2. Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling

    PubMed Central

    Tian, Xiaoxia; Yan, Huijuan; Li, Jiayi; Wu, Shuang; Wang, Junyu; Fan, Lifei

    2017-01-01

    Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5’-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins. PMID:28098758

  3. A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity.

    PubMed

    Riddick, Gregory; Kotliarova, Svetlana; Rodriguez, Virginia; Kim, H S; Linkous, Amanda; Storaska, Andrew J; Ahn, Susie; Walling, Jennifer; Belova, Galina; Fine, Howard A

    2017-03-03

    Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify.

  4. Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling.

    PubMed

    Tian, Xiaoxia; Yan, Huijuan; Li, Jiayi; Wu, Shuang; Wang, Junyu; Fan, Lifei

    2017-01-13

    Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5'-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.

  5. 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

  6. IFN-τ Alleviates Lipopolysaccharide-Induced Inflammation by Suppressing NF-κB and MAPKs Pathway Activation in Mice.

    PubMed

    Wu, Haichong; Zhao, Gan; Jiang, Kangfeng; Chen, Xiuying; Rui, Guangze; Qiu, Changwei; Guo, Mengyao; Deng, Ganzhen

    2016-06-01

    IFN-τ, which is a type I interferon with low cytotoxicity, is defined as a pregnancy recognition signal in ruminants. Type I interferons have been used as anti-inflammatory agents, but their side effects limit their clinical application. The present study aimed to determine the anti-inflammatory effects of IFN-τ in a lipopolysaccharide-stimulated acute lung injury (ALI) model and in RAW264.7 cells and to confirm the mechanism of action involved. The methods used included histopathology, measuring the lung wet/dry ratio, determining the myeloperoxidase activity, ELISA, qPCR, and western blot. The results revealed that IFN-τ greatly ameliorated the infiltration of inflammatory cells and the expression of TNF-α, IL-1β, and IL-6. Further analysis revealed that IFN-τ down-regulated the expression of TLR-2 and TLR-4 mRNA and the activity of the NF-κB and MAPK pathways both in a lipopolysaccharide-induced ALI model and in RAW264.7 cells. The results demonstrated that IFN-τ suppressed the levels of pro-inflammatory cytokines by inhibiting the phosphorylation of the NF-κB and MAPK pathways. Thus, IFN-τ may be an optimal target for the treatment of inflammatory diseases.

  7. A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity

    PubMed Central

    Riddick, Gregory; Kotliarova, Svetlana; Rodriguez, Virginia; Kim, H. S.; Linkous, Amanda; Storaska, Andrew J.; Ahn, Susie; Walling, Jennifer; Belova, Galina; Fine, Howard A.

    2017-01-01

    Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify. PMID:28256619

  8. Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

    PubMed

    Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

    2017-11-01

    Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

  9. 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

  10. IVIG inhibits TNF-α-induced MMP9 expression and activity in monocytes by suppressing NF-κB and P38 MAPK activation.

    PubMed

    Zhou, Cuizhen; Huang, Min; Xie, Lijian; Shen, Jie; Xiao, Tingting; Wang, Renjian

    2015-01-01

    Matrix metalloproteinase-9 (MMP9) has been involved in inflammatory and pathologic processes of coronary artery lesions (CAL) in Kawasaki disease (KD). Intravenous immunoglobulin (IVIG), a traditional treatment for Kawasaki disease, could decrease the expressions of MMP9. The purpose of this study was to investigate the protective effect of IVIG in chemotactic migration of monocyte and the regulation of MMP9 induced by tumor necrosis factor-α (TNF-α) in U937s. Studies were carried out with real time polymerase chain reaction (RT-PCR), zymographic, Western blotting and immunofluorescence. U937s' migration was enhanced by TNF-α stimulation, while was inhibited by IVIG pretreatment. MMP9 expression and activity in U937s were also significantly enhanced by TNF-α and inhibited by IVIVG pretreatment. During inflammatory stimulus, nuclear factor kappa B (NF-κB) and P38 Mitogenactivated protein kinase (P38 MAPK) pathways play a significant role in regulating MMP9 gene expression. TNF-α induced nuclear translocation of NF-κB and P38 MAPK activation in U937s were inhibited significantly by IVIG. Furthermore, we clarified that nuclear NF-κB and P38 MAPK pathways play pivotal roles in regulating U937s' migration and MMP9 expressions using PDTC and SB203580, which were specific inhibitors of NF-κB and p38 MAPK pathways. IVIG displays striking biological effects, notably promoting monocyte migration. These effects involve the NF-κB and p38 pathways, and increased MMP9 activity. It might be a crucial mechanism of IVIG reducing the occurrence of CAL that IVIG inhibited monocytes expressing MMP9 and decreased chemotactic migration of monocyte.

  11. Endoplasmic reticulum stress increases brain MAPK signaling, inflammation and renin-angiotensin system activity and sympathetic nerve activity in heart failure.

    PubMed

    Wei, Shun-Guang; Yu, Yang; Weiss, Robert M; Felder, Robert B

    2016-10-01

    We previously reported that endoplasmic reticulum (ER) stress is induced in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN) of heart failure (HF) rats and is reduced by inhibition of mitogen-activated protein kinase (MAPK) signaling. The present study further examined the relationship between brain MAPK signaling, ER stress, and sympathetic excitation in HF. Sham-operated (Sham) and HF rats received a 4-wk intracerebroventricular (ICV) infusion of vehicle (Veh) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10 μg/day). Lower mRNA levels of the ER stress biomarkers GRP78, ATF6, ATF4, and XBP-1s in the SFO and PVN of TUDCA-treated HF rats validated the efficacy of the TUDCA dose. The elevated levels of phosphorylated p44/42 and p38 MAPK in SFO and PVN of Veh-treated HF rats, compared with Sham rats, were significantly reduced in TUDCA-treated HF rats as shown by Western blot and immunofluorescent staining. Plasma norepinephrine levels were higher in Veh-treated HF rats, compared with Veh-treated Sham rats, and were significantly lower in the TUDCA-treated HF rats. TUDCA-treated HF rats also had lower mRNA levels for angiotensin converting enzyme, angiotensin II type 1 receptor, tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and NF-κB p65, and a higher mRNA level of IκB-α, in the SFO and PVN than Veh-treated HF rats. These data suggest that ER stress contributes to the augmented sympathetic activity in HF by inducing MAPK signaling, thereby promoting inflammation and renin-angiotensin system activity in key cardiovascular regulatory regions of the brain.

  12. Glial activation in the periaqueductal gray promotes descending facilitation of neuropathic pain through the p38 MAPK signaling pathway.

    PubMed

    Ni, Hua-Dong; Yao, Ming; Huang, Bing; Xu, Long-Sheng; Zheng, Ying; Chu, Yu-Xia; Wang, Han-Qi; Liu, Ming-Juan; Xu, Shi-Jie; Li, Hong-Bo

    2016-01-01

    The midbrain ventrolateral periaqueductal gray (VL-PAG) is a key component that mediates pain modulation. Although spinal cord glial cells appear to play an important role in chronic pain development, the precise mechanisms involving descending facilitation pathways from the PAG following nerve injury are poorly understood. This study shows that cellular events that occur during glial activation in the VL-PAG may promote descending facilitation from the PAG during neuropathic pain. Chronic constriction nerve injury (CCI) was induced by ligature construction of the sciatic nerve in male Sprague-Dawley rats. Behavioral responses to noxious mechanical (paw withdrawal threshold; PWT) and thermal (paw withdrawal latency; PWL) stimuli were evaluated. After CCI, immunohistochemical and Western blot analysis of microglia and astrocytes in the VL-PAG showed morphological and quantitative changes indicative of activation in microglia and astrocytes. Intra-VL-PAG injection of microglial or astrocytic inhibitors attenuated PWT and PWL at days 7 and 14, respectively, following CCI. We also evaluated the effects of intra-VL-PAG administration of the phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) inhibitor SB 203580 at day 7 after CCI. This treatment abolished microglial activation and produced a significant time-dependent attenuation of PWT and PWL. Western blot analysis showed localized expression of p-p38 in the VL-PAG after CCI. P-p38 was expressed in labeled microglia of the VL-PAG but was not present in astrocytes and neurons on day 7 after CCI. These results demonstrate that CCI-induced neuropathic pain is associated with glial activation in the VL-PAG, which likely participates in descending pain facilitation through the p38 MAPK signaling pathway.

  13. Modeling of the MAPK machinery activation in response to various abiotic and biotic stresses in plants by a system biology approach.

    PubMed

    Pathak, Rajesh Kumar; Taj, Gohar; Pandey, Dinesh; Arora, Sandeep; Kumar, Anil

    2013-01-01

    Mitogen-Activated Protein Kinases (MAPKs) cascade plays an important role in regulating plant growth and development, generating cellular responses to the extracellular stimuli. MAPKs cascade mainly consist of three sub-families i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK), several cascades of which are activated by various abiotic and biotic stresses. In this work we have modeled the holistic molecular mechanisms essential to MAPKs activation in response to several abiotic and biotic stresses through a system biology approach and performed its simulation studies. As extent of abiotic and biotic stresses goes on increasing, the process of cell division, cell growth and cell differentiation slow down in time dependent manner. The models developed depict the combinatorial and multicomponent signaling triggered in response to several abiotic and biotic factors. These models can be used to predict behavior of cells in event of various stresses depending on their time and exposure through activation of complex signaling cascades.

  14. The pore-forming α-toxin from clostridium septicum activates the MAPK pathway in a Ras-c-Raf-dependent and independent manner.

    PubMed

    Chakravorty, Anjana; Awad, Milena M; Cheung, Jackie K; Hiscox, Thomas J; Lyras, Dena; Rood, Julian I

    2015-02-10

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process.

  15. Transcriptional upregulation of DDR2 by ATF4 facilitates osteoblastic differentiation through p38 MAPK-mediated Runx2 activation.

    PubMed

    Lin, Kuan-Liang; Chou, Ching-Heng; Hsieh, Shu-Chen; Hwa, Su-Yang; Lee, Ming-Ta; Wang, Fung-Fang

    2010-11-01

    Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at -1150 bp in the DDR2 promoter was required for ATF4-mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast-specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2-induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation.

  16. 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.

  17. Roxatidine attenuates mast cell-mediated allergic inflammation via inhibition of NF-κB and p38 MAPK activation

    PubMed Central

    Lee, Minho; Lee, Na Young; Chung, Kyung-Sook; Cheon, Se-Yun; Lee, Kyung-Tae; An, Hyo-Jin

    2017-01-01

    Roxatidine is an active metabolite of roxatidine acetate hydrochloride which is a histamine H2-receptor antagonist that is used to treat gastric and duodenal ulcers. In this study, we investigated the anti-allergic inflammatory effects and the underlying molecular mechanism of roxatidine in phorbol 12-myristate 13-acetate and calcium ionophore (PMACI)-stimulated human mast cells-1 (HMC-1), compound 48/80-induced anaphylactic animal model and chemical allergen-induced contact hypersensitivity (CHS) models. Roxatidine suppressed the mRNA and protein expression of inflammatory cytokines such as TNF-α, IL-6, and IL-1β in PMACI-stimulated HMC-1 and compound 48/80-induced anaphylactic mice. In addition, roxatidine attenuated PMACI-induced nuclear translocation of NF-κB and the phosphorylation of MKK3/6 and MK2, which are both involved in the p38 MAPK pathway. Furthermore, we observed that roxatidine suppressed the activation of caspase-1, an IL-1β converting enzyme, in PMACI-stimulated HMC-1 and compound 48/80-induced anaphylactic mice. In CHS model, roxatidine significantly reduced ear swelling, increased number of mast cells, production levels of cytokines and migration of dendritic cells. Our findings provide evidence that the anti-allergic inflammatory properties of roxatidine are mediated by the inhibition of NF-κB and caspase-1 activation, p38 MAPK pathway and mast cell-derived cytokine production. Taken together, the in vitro and in vivo anti-allergic inflammatory effects suggest a possible therapeutic application of roxatidine in allergic inflammatory diseases. PMID:28139747

  18. MicroRNA 181b promotes vascular smooth muscle cells proliferation through activation of PI3K and MAPK pathways.

    PubMed

    Li, Tie-Jun; Chen, Yan-Li; Gua, Chao-Jun; Xue, Sheng-Jiang; Ma, Shu-Mei; Li, Xiao-Dong

    2015-01-01

    Vascular smooth muscle cells (VSMCs) hyperplasia is a common feature of pathologic cardiovascular event such as restenosis and atherosclerosis. The role and mechanisms of microRNAs (miRs) in VSMCs proliferation are poorly understood. Here, we report that miR-181b promotes VSMCs proliferation and migration. In an animal model, miR-181b was significantly increased in the rat carotid artery after balloon catheter injury. Delivery of miR-181b inhibitor to injured artery exhibited a marked inhibition of neointimal hyperplasia. Transfection of miR-181b with "mimics" to A10 cells accelerated cell proliferation, which was accompanied by an increase of cell migration. The induction of A10 cells proliferation by miR-181b appeared to be involved in activation of S and G2/M checkpoint, concomitant with decreases in cell-cycle inhibitors p21 and p27, and increases in cell-cycle activators CDK4 and cyclinD1. In contract, miR-181b inhibition attenuated A10 cells proliferation, inhibited cell migration and arrested cell cycle transition. Moreover, forced miR-181b expression elevated the phosphorylation levels of Akt and Erk1/2, whereas inhibition of miR-181b produced the opposite effects. Additionally, inhibition of PI3K and MAPK signaling pathways with specific inhibitors, but not inhibition of JNK pathway, significantly abolished the effects of miR-181b in promoting cell proliferation. These findings demonstrate that miR-181b enhances the proliferation and migration of VSMCs through activation of PI3K and MAPK pathways.

  19. Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

    PubMed Central

    Li, Zhen Hua; Zhang, Zai Jun; Hu, Guang; Cheang, Lorita Chi Veng; Alex, Deepa; Hoi, Maggie Pui Man; Kwan, Yiu Wa; Chan, Shun Wan; Leung, George Pak Heng; Lee, Simon Ming Yuen

    2010-01-01

    Background Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo. Methodology Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting. Conclusion Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and

  20. 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

  1. Activation of Nrf2 Reduces UVA-Mediated MMP-1 Upregulation via MAPK/AP-1 Signaling Cascades: The Photoprotective Effects of Sulforaphane and Hispidulin

    PubMed Central

    Chaiprasongsuk, Anyamanee; Lohakul, Jinaphat; Soontrapa, Kitipong; Sampattavanich, Somponnat; Akarasereenont, Pravit

    2017-01-01

    UVA irradiation plays a role in premature aging of the skin through triggering oxidative stress-associated stimulation of matrix metalloproteinase-1 (MMP-1) responsible for collagen degradation, a hallmark of photoaged skin. Compounds that can activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor regulating antioxidant gene expression, should therefore serve as effective antiphotoaging agents. We investigated whether genetic silencing of Nrf2 could relieve UVA-mediated MMP-1 upregulation via activation of mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) signaling using human keratinocyte cell line (HaCaT). Antiphotoaging effects of hispidulin (HPD) and sulforaphane (SFN) were assessed on their abilities to activate Nrf2 in controlling MMP-1 and collagen expressions in association with phosphorylation of MAPKs (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38), c-Jun, and c-Fos, using the skin of BALB/c mice subjected to repetitive UVA irradiation. Our findings suggested that depletion of Nrf2 promoted both mRNA expression and activity of MMP-1 in the UVA-irradiated HaCaT cells. Treatment of Nrf2 knocked-down HaCaT cells with MAPK inhibitors significantly suppressed UVA-induced MMP-1 and AP-1 activities. Moreover, pretreatment of the mouse skin with HPD and SFN, which could activate Nrf2, provided protective effects against UVA-mediated MMP-1 induction and collagen depletion in correlation with the decreased levels of phosphorylated MAPKs, c-Jun, and c-Fos in the mouse skin. In conclusion, Nrf2 could influence UVA-mediated MMP-1 upregulation through the MAPK/AP-1 signaling cascades. HPD and SFN may therefore represent promising antiphotoaging candidates. PMID:28011874

  2. FAM83B-mediated activation of PI3K/AKT and MAPK signaling cooperates to promote epithelial cell transformation and resistance to targeted therapies

    PubMed Central

    Cipriano, Rocky; Miskimen, Kristy L.S.; Bryson, Benjamin L.; Foy, Chase R.; Bartel, Courtney A.; Jackson, Mark W.

    2013-01-01

    Therapies targeting MAPK and AKT/mTOR signaling are currently being evaluated in clinical trials for several tumor types. However, recent studies suggest that these therapies may be limited due to acquired cancer cell resistance and a small therapeutic index between normal and cancer cells. The identification of novel proteins that are involved in MAPK or AKT/mTOR signaling and differentially expressed between normal and cancer cells will provide mechanistically distinct therapeutic targets with the potential to inhibit these key cancer-associated pathways. We recently identified FAM83B as a novel, previously uncharacterized oncogene capable of hyperactivating MAPK and mTOR signaling and driving the tumorigenicity of immortalized human mammary epithelial cells (HMEC). We show here that elevated FAM83B expression also activates the PI3K/AKT signaling pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR inhibitors. FAM83B co-precipitated with the p85α and p110α subunits of PI3K, as well as AKT, and increased p110α and AKT membrane localization, consistent with elevated PI3K/AKT signaling. In tumor-derived cells harboring elevated FAM83B expression, ablation of FAM83B decreased p110α and AKT membrane localization, suppressed AKT phosphorylation, and diminished proliferation, AIG, and tumorigenicity in vivo. We propose that the level of FAM83B expression may be an important factor to consider when combined therapies targeting MAPK and AKT/mTOR signaling are used. Moreover, the identification of FAM83B as a novel oncogene and its integral involvement in activating PI3K/AKT and MAPK provides a foundation for future therapies aimed at targeting FAM83B in order to suppress the growth of PI3K/AKT- and MAPK-driven cancers. PMID:23676467

  3. 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.

  4. Exendin-4 promotes proliferation and differentiation of MC3T3-E1 osteoblasts by MAPKs activation.

    PubMed

    Feng, Yingyu; Su, Lei; Zhong, Xing; Guohong, Wei; Xiao, Haipeng; Li, Yanbing; Xiu, Lingling

    2016-04-01

    Glucagon-like peptide-1 (GLP1) and its receptor agonist have been previously reported to play a positive role in bone metabolism in aged ovariectomized rats and insulin-resistant models. However, whether GLP1 has a direct effect on the proliferation and differentiation of osteoblasts or any cellular mechanism for this potential role is unknown. We examined the effects of the GLP1 receptor agonist exendin-4 on the proliferation, differentiation, and mineralization of mouse osteoblastic MC3T3-E1 cells. GLP1 receptor was detected in MC3T3-E1 cells by polymerase chain reaction (PCR) and Western blot assay. Cell proliferation was assessed using MTT assay, revealing that exendin-4 increased cell proliferation at effective concentrations between 10(-10) and 10(-5) M. Quantitative PCR analysis showed that exendin-4 increased the mRNA expression of the differentiation markers alkaline phosphatase (ALP), collagen-1 (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2) under osteogenic conditions. Alizarin red staining confirmed that 10(-7) M exendin-4 increased osteoblast mineralization by 18.7%. Exendin-4 upregulated the phosphorylation of ERK1/2, p38, and JNK, with the peak effect at 1.5 h in the Western blot analysis. The use of selective MAPK inhibitors, namely PD98059, SB203580, and SP600125, blocked the effects of exendin-4 on kinase activation (ERK1/2, p38, and JNK), as well as cell proliferation and differentiation in MC3T3-E1 cells. These findings demonstrate that exendin-4 promotes both the proliferation and differentiation of preosteoblasts MC3T3-E1 via activation of the MAPK pathway.

  5. Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination

    PubMed Central

    Sheean, Maria E.; McShane, Erik; Cheret, Cyril; Walcher, Jan; Müller, Thomas; Wulf-Goldenberg, Annika; Hoelper, Soraya; Garratt, Alistair N.; Krüger, Markus; Rajewsky, Klaus; Meijer, Dies; Birchmeier, Walter; Lewin, Gary R.; Selbach, Matthias; Birchmeier, Carmen

    2014-01-01

    Myelination depends on the synthesis of large amounts of myelin transcripts and proteins and is controlled by Nrg1/ErbB/Shp2 signaling. We developed a novel pulse labeling strategy based on stable isotope labeling with amino acids in cell culture (SILAC) to measure the dynamics of myelin protein production in mice. We found that protein synthesis is dampened in the maturing postnatal peripheral nervous system, and myelination then slows down. Remarkably, sustained activation of MAPK signaling by expression of the Mek1DD allele in mice overcomes the signals that end myelination, resulting in continuous myelin growth. MAPK activation leads to minor changes in transcript levels but massively up-regulates protein production. Pharmacological interference in vivo demonstrates that the effects of activated MAPK signaling on translation are mediated by mTOR-independent mechanisms but in part also by mTOR-dependent mechanisms. Previous work demonstrated that loss of ErbB3/Shp2 signaling impairs Schwann cell development and disrupts the myelination program. We found that activated MAPK signaling strikingly compensates for the absence of ErbB3 or Shp2 during Schwann cell development and myelination. PMID:24493648

  6. Non-thermal activation of the hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer- and blood-brain barrier-related effects.

    PubMed

    Leszczynski, Dariusz; Joenväärä, Sakari; Reivinen, Jukka; Kuokka, Reetta

    2002-05-01

    We have examined whether non-thermal exposures of cultures of the human endothelial cell line EA.hy926 to 900 MHz GSM mobile phone microwave radiation could activate stress response. Results obtained demonstrate that 1-hour non-thermal exposure of EA.hy926 cells changes the phosphorylation status of numerous, yet largely unidentified, proteins. One of the affected proteins was identified as heat shock protein-27 (hsp27). Mobile phone exposure caused a transient increase in phosphorylation of hsp27, an effect which was prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38MAPK). Also, mobile phone exposure caused transient changes in the protein expression levels of hsp27 and p38MAPK. All these changes were non-thermal effects because, as determined using temperature probes, irradiation did not alter the temperature of cell cultures, which remained throughout the irradiation period at 37 +/- 0.3 degrees C. Changes in the overall pattern of protein phosphorylation suggest that mobile phone radiation activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK stress response pathway. Based on the known functions of hsp27, we put forward the hypothesis that mobile phone radiation-induced activation of hsp27 may (i) facilitate the development of brain cancer by inhibiting the cytochrome c/caspase-3 apoptotic pathway and (ii) cause an increase in blood-brain barrier permeability through stabilization of endothelial cell stress fibers. We postulate that these events, when occurring repeatedly over a long period of time, might become a health hazard because of the possible accumulation of brain tissue damage. Furthermore, our hypothesis suggests that other brain damaging factors may co-participate in mobile phone radiation-induced effects.

  7. Dentate Gyrus Development Requires ERK Activity to Maintain Progenitor Population and MAPK Pathway Feedback Regulation

    PubMed Central

    Vithayathil, Joseph; Pucilowska, Joanna; Goodnough, L. Henry; Atit, Radhika P.

    2015-01-01

    The ERK/MAPK pathway is an important developmental signaling pathway. Mutations in upstream elements of this pathway result in neuro-cardio-facial cutaneous (NCFC) syndromes, which are typified by impaired neurocognitive abilities that are reliant upon hippocampal function. The role of ERK signaling during hippocampal development has not been examined and may provide critical insight into the cause of hippocampal dysfunction in NCFC syndromes. In this study, we have generated ERK1 and conditional ERK2 compound knock-out mice to determine the role of ERK signaling during development of the hippocampal dentate gyrus. We found that loss of both ERK1 and ERK2 resulted in 60% fewer granule cells and near complete absence of neural progenitor pools in the postnatal dentate gyrus. Loss of ERK1/2 impaired maintenance of neural progenitors as they migrate from the dentate ventricular zone to the dentate gyrus proper, resulting in premature depletion of neural progenitor cells beginning at E16.5, which prevented generation of granule cells later in development. Finally, loss of ERK2 alone does not impair development of the dentate gyrus as animals expressing only ERK1 developed a normal hippocampus. These findings establish that ERK signaling regulates maintenance of progenitor cells required for development of the dentate gyrus. PMID:25926459

  8. 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.

  9. Persistent mechanical stretch-induced calcium overload and MAPK signal activation contributed to SCF reduction in colonic smooth muscle in vivo and in vitro.

    PubMed

    Dong, Fang; Yang, Shu; Sun, Haimei; Yan, Jihong; Guo, Xiaoxia; Li, Dandan; Zhou, Deshan

    2017-04-01

    Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca(2+) overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca(2+) overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca(2+) overload was originated from extracellular Ca(2+) influx and endoplasmic reticulum (ER) Ca(2+) release identified by treating with different Ca(2+) channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca(2+) overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.

  10. 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

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

    PubMed

    Occhi, Gianluca; Barollo, Susi; 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

    2015-10-13

    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.

  12. 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.

  13. 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.

  14. 7-O-Geranylquercetin induces apoptosis in gastric cancer cells via ROS-MAPK mediated mitochondrial signaling pathway activation.

    PubMed

    Zhu, Yanyan; Jiang, Yameng; Shi, Lei; Du, Linying; Xu, Xiaodong; Wang, Enxia; Sun, Yong; Guo, Xin; Zou, Boyang; Wang, Huaxin; Wang, Changyuan; Sun, Lidan; Zhen, Yuhong

    2017-03-01

    7-O-Geranylquercetin (GQ) is a novel O-alkylated derivate of quercetin. In this study, we evaluated its apoptosis induction effects in human gastric cancer cell lines SGC-7901 and MGC-803 and explored the potential molecular mechanisms. The results demonstrated that GQ lowered viability of SGC-7901 and MGC-803 cells in a dose- and time-dependent manner without apparent cytotoxicity to human gastric epithelial cell line GES-1. GQ could induce apoptosis in SGC-7901 and MGC-803cells, and arrest the gastric cancer cells at G2/M phase. Mechanism study showed that GQ triggered generation of reactive oxygen species (ROS), then activated p38 and JNK signaling pathways, subsequently led to mitochondrial impairment by regulating the expression of Bcl-2, Bcl-xl and Bax, and finally promoted the release of cytochrome c and the activation of caspases to induce apoptosis. In addition, Z-VAD-FMK (caspase inhibitor) could reverse GQ-induced apoptosis. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) could rescue GQ-induced cell death and attenuate mitochondrial signal pathway activation. Furthermore, NAC (ROS inhibitor) could rescue GQ-induced cell death, reduce ROS generation, decrease the phosphorylation of p38 and JNK, and then attenuate the activation of mitochondrial signal pathway. Taken together, GQ induces caspase-dependent apoptosis in gastric cancer cells through activating ROS-MAPK mediated mitochondrial signal pathway. This study highlights the potential use of GQ as a gastric cancer therapeutic agent.

  15. The Ethanol Extract of Fructus trichosanthis Promotes Fetal Hemoglobin Production via p38 MAPK Activation and ERK Inactivation in K562 Cells

    PubMed Central

    Li, Hui; Ko, Chun Hay; Tsang, Suk Ying; Leung, Ping Chung; Fung, Ming Chui; Fung, Kwok Pui

    2011-01-01

    Pharmacological stimulation of fetal hemoglobin (HbF) expression may be a promising approach for the treatment of beta-thalassemia. In this study, the effects of Fructus trichosanthis (FT) were investigated in human erythroleukemic K562 cells for their gamma-globin mRNA and HbF-induction activities. The role of signaling pathways, including extracellular regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), was also investigated. It was found that the ethanol extract of FT significantly increased gamma-globin mRNA and HbF levels, determined by real-time reverse transcription polymerase chain reaction and enzyme linked immunosorbent assay, respectively, in dose- and time-dependent manner. Total Hb (THb) levels were also elevated in the concentrations without cytotoxicity (<80 μg mL−1). Pre-treatment with p38 MAPK inhibitor SB203580 blocked the stimulatory effects of FT extract in total and HbF induction. In contrast, no change in HbF was observed when treated with ERK inhibitor PD98059. Furthermore, FT ethanol extract activated p38 MAPK and inhibited ERK signaling pathways in K562 cells, as revealed in western blotting analysis. In addition, SB203580 significantly abolished p38 MAPK activation when the cells were treated with FT. In summary, the ethanol extract of FT was found to be a potent inducer of HbF synthesis in K562 cells. The present data delineated the role of ERK and p38 MAPK signaling as molecular targets for pharmacologic stimulation of HbF production upon FT treatment. PMID:21876711

  16. Effects of budesonide on P38 MAPK activation, apoptosis and IL-8 secretion, induced by TNF-alpha and Haemophilus influenzae in human bronchial epithelial cells.

    PubMed

    Gallelli, L; Pelaia, G; Fratto, D; Muto, V; Falcone, D; Vatrella, A; Curto, L S; Renda, T; Busceti, M T; Liberto, M C; Savino, R; Cazzola, M; Marsico, S A; Maselli, R

    2010-01-01

    Non-typeable Haemophilus influenzae (NTHi) is one of the most frequently involved pathogens in bacterial exacerbations of chronic obstructive pulmonary disease (COPD). In the airways, the main tissue target of NTHi is bronchial epithelium, where this pathogen can further amplify the inflammatory and structural changes induced by proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha). Therefore, the aim of this study is to investigate, in primary cultures of human bronchial epithelial cells, the effects of NTHi on signal transduction pathways, apoptotic events and chemokine production activated by TNF-alpha. Moreover, we also evaluated the effects exerted on such cellular and molecular phenomena by a corticosteroid drug. p38 mitogen-activated protein kinase (MAPK) phosphorylation was analyzed by Western blotting, using an anti-phospho-p38 MAPK monoclonal antibody. Apoptosis was assayed by active caspase-3 expression. Interleukin-8 (IL-8/CXCL8) was detected in cell-free culture supernatants by ELISA. TNF-alpha induced a significant increase in p38 MAPK phosphorylation. NTHi was able to potentiate the stimulatory actions of TNF-alpha on caspase-3 expression and, to a lesser extent, on IL-8 secretion. These effects were significantly (P less than 0.01) inhibited by a pharmacological pre-treatment with budesonide. These results suggest that TNF-alpha is able to stimulate, via activation of p38 MAPK signalling pathway, IL-8 release and airway epithelial cell apoptosis; the latter effect can be markedly potentiated by NTHi. Furthermore, budesonide can be very effective in preventing, through inhibition of p38 MAPK phosphorylation, both structural and proinflammatory changes elicited in bronchial epithelium by TNF-alpha and NTHi.

  17. 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

  18. 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

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

    2016-10-01

    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 ribonuclease (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.

  19. Decitabine and 5-azacitidine both alleviate LPS induced ARDS through anti-inflammatory/antioxidant activity and protection of glycocalyx and inhibition of MAPK pathways in mice.

    PubMed

    Huang, Xiao; Kong, Guiqing; Li, Yan; Zhu, Weiwei; Xu, Haixiao; Zhang, Xiaohua; Li, Jiankui; Wang, Lipeng; Zhang, Zhongwen; Wu, Yaru; Liu, Xiangyong; Wang, Xiaozhi

    2016-12-01

    Decitabine (5-aza-2'-deoxycytidine, DAC) and 5-azacitidine (Aza), an inhibitor of DNA methyltransferases, possess a wide range of anti-metabolic and anti-cancer activities. This study examined the effects of DAC and Aza on inflammatory and oxidative injuries, as well as on glycocalyx and MAPK signaling pathways, in a LPS-stimulated ARDS mouse model. Results of ELISA revealed that DAC and Aza significantly inhibited the production of TNF-α and IL-1β and prevented LPS-induced elevation of myeloperoxidase and malondialdehyde levels in serum. The W/D ratio of lung and histopathologic examination with hematoxylin and eosin staining showed that DAC and Aza pretreatment substantially improved lung tissue injury. DAC and Aza reduced the level of glycocalyx degradation products (e.g., heparan sulfate and haluronic acid) and protected glycocalyx integrity. Western blot assay demonstrated that DAC and Aza both significantly suppressed LPS-induced activation of the MAPK signaling pathways by blocking the phosphorylation of JNK, ERK and P38 in lung tissues. Bisulfite sequencing PCR and real time-PCR showed that DAC reversed the RASSF1A promoter hypermethylation and furthermore elevated the expression of RASSF1A, which is a tumor suppressor that regulates MAPK signaling pathway. These results suggested that DAC inhibited the MAPK signaling pathway in LPS-induced ARDS mice might via demethylation in RASSF1A promoter region and by restoring its expression. This study highlighted the close relationship between DNA methylation and the development and progression of ARDS.

  20. 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

  1. Mitogen-activated Protein Kinase (MAPK) Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress*

    PubMed Central

    Riquelme, Manuel A.; Burra, Sirisha; Kar, Rekha; Lampe, Paul D.; Jiang, Jean X.

    2015-01-01

    Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however, sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at Ser279-Ser282, sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pretreated with the MAPK pathway inhibitor. We found that prostaglandin E2 (PGE2) accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at Ser279-Ser282 sites. Depletion of PGE2 from CM, and pre-treatment with a p44/42 ERK pathway-specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. A similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure. PMID:26442583

  2. 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

  3. Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains.

    PubMed

    Rimoldi, Valeria; Reversi, Alessandra; Taverna, Elena; Rosa, Patrizia; Francolini, Maura; Cassoni, Paola; Parenti, Marco; Chini, Bice

    2003-09-04

    We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21(WAF1/CIP1), thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.

  4. 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

  5. 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.

  6. Polysaccharides from Capsosiphon fulvescens stimulate the growth of IEC-6 Cells by activating the MAPK signaling pathway.

    PubMed

    Go, Hiroe; Hwang, Hye-Jung; Nam, Taek-Jeong

    2011-06-01

    Seaweed extracts show diverse bioactivities, such as antioxidant and antitumor activity. Capsosiphon fulvescens is a green alga that is abundant along the southwest coast of South Korea. Although it is consumed for its purported health-enhancing properties, particularly as a treatment for stomach disorders and hangovers, the health effects of dietary C. fulvescens remain unclear. We extracted polysaccharides from C. fulvescens (Cf-PS), investigated their effects on the proliferation of rat small intestinal epithelial IEC-6 cells, and determined the signaling cascade involved. We cultured IEC-6 cells in the presence of Cf-PS, which stimulated cell proliferation in a dose-dependent manner, and analyzed the Wnt and MAPK signaling pathways, which are related to cell proliferation. Cf-PS treatment induced the translocation of β-catenin, an effector of the Wnt signaling pathway, from the cytosol to the nucleus and increased the expression of cyclinD1 and c-myc. Cf-PS also induced ERK1/2 phosphorylation, which is activated by mitogenic and proliferative stimuli such as growth factors, but the phosphorylation of JNK and p38 was not enhanced. Our results show that Cf-PS regulates proliferation via stimulating the nuclear translocation of β-catenin and ERK1/2 activation in intestinal epithelial cells.

  7. Galangin induces B16F10 melanoma cell apoptosis via mitochondrial pathway and sustained activation of p38 MAPK.

    PubMed

    Zhang, Wenjing; Lan, Yan; Huang, Qilai; Hua, Zichun

    2013-05-01

    Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent.

  8. 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.

  9. Tyrosine phosphorylation of Kir3 following kappa-opioid receptor activation of p38 MAPK causes heterologous desensitization.

    PubMed

    Clayton, Cecilea C; Xu, Mei; Chavkin, Charles

    2009-11-13

    Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K(ir)3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K(ir)3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wild-type mice, but not in mice lacking the kappa-opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K(ir)3.1-ir. The U50,488-induced increase in pY12-K(ir)3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K(ir)3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K(ir)3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K(ir)3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K(ir)3.1-ir; and the heterologous desensitization of K(ir)3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K(ir)3.1 and channel inhibition through a GRK3-, p38 MAPK- and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.

  10. Shengmai Formula suppressed over-activated Ras/MAPK pathway in C. elegans by opening mitochondrial permeability transition pore via regulating cyclophilin D

    PubMed Central

    Liu, Yan; Zhi, Dejuan; Li, Menghui; Liu, Dongling; Wang, Xin; Wu, Zhengrong; Zhang, Zhanxin; Fei, Dongqing; Li, Yang; Zhu, Hongmei; Xie, Qingjian; Yang, Hui; Li, Hongyu

    2016-01-01

    Since about 30% of all human cancers contain mutationally activated Ras, down regulating the over-activation of Ras/MAPK pathway represents a viable approach for treating cancers. Over-activation of Ras/MAPK pathway is accompanied by accumulation of reactive oxygen species (ROS). One approach for developing anti-cancer drugs is to target ROS production and their accumulation. To test this idea, we have employed C. elegans of let-60 (gf) mutant, which contain over-activated let-60 (the homolog of mammalian ras) and exhibit tumor-like symptom of multivulva phenotype, to determine whether anti-oxidants can affect their tumor-like phenotype. Specifically we studied the effect of Shengmai formula (SM), a traditional Chinese medicine that has strong anti-oxidant activity, on the physiology of let-60 (gf) mutants. Unexpectedly, we found that SM treatment led to the opening of mitochondrial permeability transition pore by regulating cyclophilin D and then triggered oxidative stress and related signaling pathway activation, including p53, JNK, and p38/MAPK pathways. Finally, SM induced mitochondrial pathway of apoptosis and inhibited the tumor-like symptom of the multivulva phenotype of let-60(gf) mutants. Our results provide evidences to support that SM act as a pro-oxidant agent and could serve as a potential drug candidate for combating over-activated Ras-related cancer. PMID:27982058

  11. p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).

    PubMed

    Di Cristofano, A; Niki, M; Zhao, M; Karnell, F G; Clarkson, B; Pear, W S; Van Aelst, L; Pandolfi, P P

    2001-08-06

    p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).

  12. Rho1 GTPase and PKC ortholog Pck1 are upstream activators of the cell integrity MAPK pathway in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Madrid, Marisa; Viana, Raúl A; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.

  13. Subanesthetic Isoflurane Reduces Zymosan-Induced Inflammation in Murine Kupffer Cells by Inhibiting ROS-Activated p38 MAPK/NF-κB Signaling

    PubMed Central

    Wang, Hui; Wang, Lei; Li, Nan-lin; Li, Jun-tang; Yu, Feng; Zhao, Ya-li; Wang, Ling; Yi, Jun; Wang, Ling; Bian, Jie-fang; Chen, Jiang-hao; Yuan, Shi-fang; Wang, Ting; Lv, Yong-gang; Liu, Ning-ning; Zhu, Xiao-shan; Ling, Rui; Yun, Jun

    2014-01-01

    Volatile anesthetic isoflurane (ISO) has immunomodulatory effects. The fungal component zymosan (ZY) induces inflammation through toll-like receptor 2 or dectin-1 signaling. We investigated the molecular actions of subanesthetic (0.7%) ISO against ZY-induced inflammatory activation in murine Kupffer cells (KCs), which are known as the resident macrophages within the liver. We observed that ISO reduced ZY-induced cyclooxygenase 2 upregulation and prostaglandin E2 release, as determined by western blot and radioimmunoassay, respectively. ISO also reduced the production of tumor necrosis factor-α, interleukin-1β, IL-6, high-mobility group box-1, macrophage inflammatory protein-1α, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 as assessed by enzyme-linked immunosorbent assays. ISO blocked the ZY-induced nuclear translocation and DNA-binding activity of nuclear factor- (NF)-κB p65. Moreover, ISO attenuated ZY-induced p38 mitogen-activated protein kinase (MAPK) activation partly by scavenging reactive oxygen species (ROS); the interregulation that ROS activated p38 MAPK followed by NF-κB activation was crucial for the ZY-induced inflammatory responses in KCs. An in vivo study by peritoneal injection of ZY into BALB/C mice confirmed the anti-inflammatory properties of 0.7% ISO against ZY in KCs. These results suggest that ISO ameliorates ZY-induced inflammatory responses in murine KCs by inhibiting the interconnected ROS/p38 MAPK/NF-κB signaling pathways. PMID:25147596

  14. Long non-coding RNA Malat1 promotes neurite outgrowth through activation of ERK/MAPK signalling pathway in N2a cells.

    PubMed

    Chen, Lei; Feng, Peimin; Zhu, Xi; He, Shixu; Duan, Jialan; Zhou, Dong

    2016-11-01

    Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are playing critical roles in neurogenesis, yet the underlying molecular mechanisms remain largely elusive. Neurite outgrowth is an early step in neuronal differentiation and regeneration. Using in vitro differentiation of neuroblastoma-derived Neuro-2a (N2a) cell as a model, we performed expression profiling to identify lncRNAs putatively relevant for neurite outgrowth. We identified that Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was one of the most significantly up-regulated lncRNAs during N2a cell differentiation. Malat1 knockdown resulted in defects in neurite outgrowth as well as enhanced cell death. To pinpoint signalling pathways perturbed by Malat1 depletion, we then performed a reporter-based screening to examine the activities of 50 signalling pathways in Malat1 knockdown cells. We found that Malat1 knockdown resulted in conspicuous inhibition of Mitogen-Activated Protein Kinase (MAPK) signaling pathway as well as abnormal activation of Peroxisome proliferator-activated receptor (PPAR) and P53 signalling pathway. Inhibition of ERK/MAPK pathway with PD98059 potently blocked N2a cell neurite outgrowth, whereas phorbol 12-myristate 13-acetate-induced ERK activation rescued defects in neurite outgrowth and cell death induced by Malat1 depletion. Together, our results established a critical role of Malat1 in the early step of neuronal differentiation through activating ERK/MAPK signalling pathway.

  15. Protein O-fucosyltransferase 1 promotes trophoblast cell proliferation through activation of MAPK and PI3K/Akt signaling pathways.

    PubMed

    Liu, Chang; Liang, Xiaohua; Wang, Jiao; Zheng, Qin; Zhao, Yue; Khan, Muhammad Noman; Liu, Shuai; Yan, Qiu

    2017-04-01

    Protein O-fucosylation is an important glycosylation modification and plays an important role in embryonic development. Protein O-fucosyltransferase 1 (poFUT1) is an essential enzyme that catalyzes the synthesis of protein O-fucosylation. Our previous studies showed that poFUT1 promoted trophoblast cell migration and invasion at the fetal-maternal interface, but the role of poFUT1 in trophoblast cells proliferation remains unclear. Here, immunohistochemistry data showed that poFUT1 and PCNA levels were decreased in abortion patient's trophoblasts compared with women with normal pregnancies. Our results also showed that poFUT1 promoted trophoblast cell proliferation by CCK-8 assay and cell cycle analysis. PoFUT1 increased the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt, while inhibitors of ERK1/2(PD98059), p38 MAPK(SB203580), and PI3K (LY294002) prevented ERK1/2, p38 MAPK, and Akt phosphorylation. Moreover, poFUT1 stimulation of trophoblast cells proliferation correlated with increased cell cycle progression by promoting cells into S-phase. The underlying mechanism involved increased cyclin D1, cyclin E, CDK 2, CDK 4, and pRb expression and decreased levels of the cyclin-dependent kinase inhibitors p21 and p27, which were blocked by inhibitors of the upstream signaling molecules MAPK and PI3K/Akt. In conclusion, poFUT1 promotes trophoblast cell proliferation by activating MAPK and PI3K/Akt signaling pathways.

  16. 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.

  17. 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

  18. Rhesus macaque θ-defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF-κB and MAPK pathways

    PubMed Central

    Tongaonkar, Prasad; Trinh, Katie K.; Schaal, Justin B.; Tran, Dat; Gulko, Percio S.; Ouellette, André J.; Selsted, Michael E.

    2015-01-01

    θ-Defensins are pleiotropic, macrocyclic peptides that are expressed uniquely in Old World monkeys. The peptides are potent, broad-spectrum microbicides that also modulate inflammatory responses in vitro and in animal models of viral infection and polymicrobial sepsis. θ-Defensins suppress proinflammatory cytokine secretion by leukocytes stimulated with diverse Toll-like receptor (TLR) ligands. Studies were performed to delineate anti-inflammatory mechanisms of rhesus θ-defensin 1 (RTD-1), the most abundant θ-defensin isoform in macaque granulocytes. RTD-1 reduced the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-8 in lipopolysaccharide (LPS)-stimulated human blood monocytes and THP-1 macrophages, and this was accompanied by inhibition of nuclear factor κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) pathways. Peptide inhibition of NF-κB activation occurred following stimulation of extracellular (TLRs 1/2 and 4) and intracellular (TLR9) receptors. Although RTD-1 did not inhibit MAPK in unstimulated cells, it induced phosphorylation of Akt in otherwise untreated monocytes and THP-1 cells. In the latter, this occurred within 10 min of RTD-1 treatment and produced a sustained elevation of phosphorylated Akt (pAkt) for at least 4 h. pAkt is a negative regulator of MAPK and NF-κB activation. RTD-1 inhibited IκBα degradation and p38 MAPK phosphorylation, and stimulated Akt phosphorylation in LPS-treated human primary monocytes and THP-1 macrophages. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) blocked RTD-1-stimulated Akt phosphorylation and reversed the suppression of NF-κB activation by the peptide. These studies indicate that the anti-inflammatory properties of θ-defensins are mediated by activation of the PI3K/Akt pathway and suppression of proinflammatory signals in immune-stimulated cells. PMID:26269197

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

    PubMed

    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, PGE2, LTB4 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.

  20. ROS-mediated JNK/p38-MAPK activation regulates Bax translocation in Sorafenib-induced apoptosis of EBV-transformed B cells.

    PubMed

    Park, Ga Bin; Choi, Yunock; Kim, Yeong Seok; Lee, Hyun-Kyung; Kim, Daejin; Hur, Dae Young

    2014-03-01

    Sorafenib (SRF) is a multi-kinase inhibitor that has been shown to have antitumor activity against several types of cancers, but the effect of SRF on EBV-transformed B cells is unknown. We report that SRF can induce the apoptosis of EBV-transformed B cells through JNK/p38-MAPK activation. SRF triggered the generation of reactive oxygen species (ROS), translocation of Bax into the mitochondria, disruption of mitochondrial membrane potential, activation of caspase-9, caspase-3 and PARP, and subsequent apoptosis. Moreover, we found that SRF exposure activated the phosphorylation of JNK and p38-MAPK and suppressed the phosphorylation of PI3K-p85 and Akt. N-acetyl-l-cysteine (NAC) inhibited the activation of JNK and p38-MAPK. SP600125 and SB203580 blocked apoptosis and mitochondrial membrane disruption but did not affect ROS production after SRF treatment. These findings provide novel insights into the molecular mechanisms driving SRF-mediated cell death and suggest that SRF could be a potential therapeutic drug for the treatment of EBV-related malignant diseases.

  1. 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

    2015-10-05

    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.

  2. Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways.

    PubMed

    Stapleton, Cliona M; Joo, Joung Hyuck; Kim, Yong-Sik; Liao, Grace; Panettieri, Reynold A; Jetten, Anton M

    2010-02-15

    In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor alpha are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCalpha. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.

  3. Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells.

    PubMed

    Regan, Andrew D; Cohen, Rebecca D; Whittaker, Gary R

    2009-02-05

    Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.

  4. Berberine differentially modulates the activities of ERK, p38 MAPK, and JNK to suppress Th17 and Th1 T cell differentiation in type 1 diabetic mice.

    PubMed

    Cui, Guoliang; Qin, Xia; Zhang, Yuebo; Gong, Zhenwei; Ge, Baoxue; Zang, Ying Qin

    2009-10-09

    Berberine, an alkaloid derivative from Berberis vulgaris L., has been used extensively in traditional Chinese medicine to treat diarrhea and diabetes, but the underlying mechanisms for treating diabetes are not fully understood. Recent studies suggested that berberine has many beneficial biological effects, including anti-inflammation. Because type 1 diabetes is caused by T cell-mediated destruction of beta cells and severe islet inflammation, we hypothesized that berberine could ameliorate type 1 diabetes through its immune regulation properties. Here we reported that 2 weeks of oral administration of berberine prevented the progression of type 1 diabetes in half of the NOD mice and decreased Th17 and Th1 cytokine secretion. Berberine suppressed Th17 and Th1 differentiation by reducing the expression of lineage markers. We found that berberine inhibited Th17 differentiation by activating ERK1/2 and inhibited Th1 differentiation by inhibiting p38 MAPK and JNK activation. Berberine down-regulated the activity of STAT1 and STAT4 through the suppression of p38 MAPK and JNK activation, and it controlled the stability of STAT4 through the ubiquitin-proteasome pathway. Our findings indicate that berberine targets MAPK to suppress Th17 and Th1 differentiation in type 1 diabetic NOD mice. This study revealed a novel role of ERK in Th17 differentiation through down-regulation of STAT3 phosphorylation and RORgamma t expression.

  5. Cardiotonic pill attenuates white matter and hippocampal damage via inhibiting microglial activation and downregulating ERK and p38 MAPK signaling in chronic cerebral hypoperfused rat

    PubMed Central

    2013-01-01

    Background The cardiotonic pill (CP) is a herbal medicine composed of Salvia miltiorrhiza (SM), Panax notoginseng (PN), and Dryobalanops aromatica Gaertner (DAG) that is widely used to treat cardiovascular diseases. The present experiment was conducted to examine the effects of CP on white matter and hippocampal damage induced by chronic cerebral hypoperfusion. Methods Chronic cerebral hypoperfusion was induced in male Wistar rats by permanent bilateral common carotid artery occlusion (BCCAo). Daily oral administration of CP (200 mg/kg) began 21 days after BCCAo and continued for 42 days. The levels of microglial activation and myelin basic protein (MBP) were measured in the white matter and hippocampus of rats with chronic BCCAo, and the expression levels of mitogen-activated protein kinases (MAPKs) and inflammatory markers such as cyclooxygenase-2, interleukin-1β, and interleukin-6 were examined. Results MBP expression was reduced in the white matter and hippocampal regions of rats that received BCCAo. In contrast, reduced levels of MBP were not observed in BCCAo rats given CP treatments. The administration of CP alleviated microglial activation, the alteration of ERK and p38 MAPK signaling, and inflammatory mediator expression in rats with chronic BCCAo. Conclusion These results suggest that CP may have protective effects against chronic BCCAo-induced white matter and hippocampal damage by inhibiting inflammatory processes including microglial activation and proinflammatory mediator expression, and downreguating the hyperphosphorylation of ERK and p38 MAPK signaling. PMID:24274593

  6. Chebulagic acid inhibits the LPS-induced expression of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation.

    PubMed

    Liu, Yueying; Bao, Luer; Xuan, Liying; Song, Baohua; Lin, Lin; Han, Hao

    2015-07-01

    Inflammatory response in the vasculature, including the overexpression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, has been demonstrated to increase the risk of thrombosis development. Chebulagic acid (CA) is a key chemical component in the traditional Mongolian anti-thrombotic drug Garidi-13, and has been suggested to exert anti-inflammatory and anti-infective effects. The present study aimed to evaluate the regulatory impact of CA on a number of biological processes, including lipopolysaccharide (LPS)-induced inflammation, LPS-promoted mitogen-activated protein kinase (MAPK) activation and the expression of toll-like receptor (TLR)4 in EA.hy926 human endothelial cells. The results indicated that CA significantly inhibited the LPS-induced upregulation of TNF-α and IL-1β in a dose- and time-dependent manner. Furthermore, LPS-activated MAPK signaling was inhibited by CA treatment in the EA.hy926 cells. However, TLR4, which serves a key function in LPS-induced inflammation as the receptor of LPS, was not regulated by the CA treatment. In summary, the results of the present study indicate that CA inhibits the LPS-induced promotion of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation, which may contribute to the anti-thrombotic effect of Garidi-13.

  7. H2O2 preconditioning modulates phase II enzymes through p38 MAPK and PI3K/Akt activation.

    PubMed

    Angeloni, Cristina; Motori, Elisa; Fabbri, Daniele; Malaguti, Marco; Leoncini, Emanuela; Lorenzini, Antonello; Hrelia, Silvana

    2011-06-01

    Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules and occurs in a biphasic pattern: an early phase after 1-2 h and a late phase after 12-24 h. While it is widely accepted that reactive oxygen species are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H(2)O(2)-induced cardioprotection in rat neonatal cardiomyocytes. We focused on antioxidant and phase II enzymes and their modulation by protein kinase signaling pathways and nuclear-factor-E(2)-related factor-1 (Nrf1) and Nrf2. H(2)O(2) preconditioning was able to counteract oxidative stress more effectively in the late than in the early phase of adaptation. In particular, H(2)O(2) preconditioning counteracted oxidative stress-induced apoptosis by decreasing caspase-3 activity, increasing Bcl2 expression and selectively increasing the expression and activity of antioxidant and phase II enzymes through Nrf1 and Nrf2 translocation to the nucleus. The downregulation of Nrf1 and Nrf2 by small interfering RNA reduced the expression level of phase II enzymes. Specific inhibitors of phosphatidylinositol 3-kinase/Akt and p38 MAPK activation partially reduced the cardioprotection elicited by H(2)O(2) preconditioning and the induction and activity of phase II enzymes. These findings demonstrate, for the first time, a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes triggered by H(2)O(2) preconditioning.

  8. Hypoglycemic Effect of Opuntia ficus-indica var. saboten Is Due to Enhanced Peripheral Glucose Uptake through Activation of AMPK/p38 MAPK Pathway.

    PubMed

    Leem, Kang-Hyun; Kim, Myung-Gyou; Hahm, Young-Tae; Kim, Hye Kyung

    2016-12-09

    Opuntia ficus-indica var. saboten (OFS) has been used in traditional medicine for centuries to treat several illnesses, including diabetes. However, detailed mechanisms underlying hypoglycemic effects remain unclear. In this study, the mechanism underlying the hypoglycemic activity of OFS was evaluated using in vitro and in vivo systems. OFS treatment inhibited α-glucosidase activity and intestinal glucose absorption assessed by Na⁺-dependent glucose uptake using brush border membrane vesicles. AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle, and p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. In the present study, OFS dose-dependently increased glucose uptake in L6 muscle cells. The AMPK and p38 MAPK phosphorylations were stimulated by OFS, and inhibitors of AMPK (compound C) and p38 MAPK (SB203580) abolished the effects of OFS. Furthermore, OFS increased glucose transporter 4 (GLUT4) translocation to the plasma membrane. OFS administration (1 g/kg and 2 g/kg body weight) in db/db mice dose-dependently ameliorated hyperglycemia, hyperinsulinemia, and glucose tolerance. Insulin resistance assessed by homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index were also dose-dependently improved with OFS treatment. OFS administration improved pancreatic function through increased β-cell mass in db/db mice. These findings suggest that OFS acts by inhibiting glucose absorption from the intestine and enhancing glucose uptake from insulin-sensitive muscle cells through the AMPK/p38 MAPK signaling pathway.

  9. Hypoglycemic Effect of Opuntia ficus-indica var. saboten Is Due to Enhanced Peripheral Glucose Uptake through Activation of AMPK/p38 MAPK Pathway

    PubMed Central

    Leem, Kang-Hyun; Kim, Myung-Gyou; Hahm, Young-Tae; Kim, Hye Kyung

    2016-01-01

    Opuntia ficus-indica var. saboten (OFS) has been used in traditional medicine for centuries to treat several illnesses, including diabetes. However, detailed mechanisms underlying hypoglycemic effects remain unclear. In this study, the mechanism underlying the hypoglycemic activity of OFS was evaluated using in vitro and in vivo systems. OFS treatment inhibited α-glucosidase activity and intestinal glucose absorption assessed by Na+-dependent glucose uptake using brush border membrane vesicles. AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle, and p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. In the present study, OFS dose-dependently increased glucose uptake in L6 muscle cells. The AMPK and p38 MAPK phosphorylations were stimulated by OFS, and inhibitors of AMPK (compound C) and p38 MAPK (SB203580) abolished the effects of OFS. Furthermore, OFS increased glucose transporter 4 (GLUT4) translocation to the plasma membrane. OFS administration (1 g/kg and 2 g/kg body weight) in db/db mice dose-dependently ameliorated hyperglycemia, hyperinsulinemia, and glucose tolerance. Insulin resistance assessed by homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index were also dose-dependently improved with OFS treatment. OFS administration improved pancreatic function through increased β-cell mass in db/db mice. These findings suggest that OFS acts by inhibiting glucose absorption from the intestine and enhancing glucose uptake from insulin-sensitive muscle cells through the AMPK/p38 MAPK signaling pathway. PMID:27941667

  10. Genistein regulates the IL-1 beta induced activation of MAPKs in human periodontal ligament cells through G protein-coupled receptor 30.

    PubMed

    Luo, Li-Jun; Liu, Feng; Lin, Zhi-Kai; Xie, Yu-Feng; Xu, Jia-Li; Tong, Qing-Chun; Shu, Rong

    2012-06-01

    Periodontal ligament (PDL) cells are fibroblasts that play key roles in tissue integrity, periodontal inflammation and tissue regeneration in the periodontium. The periodontal tissue destruction in periodontitis is mediated by host tissue-produced inflammatory cytokines, including interleukin-1β (IL-1β). Here, we report the expression of G protein-coupled receptor 30 (GPR30, also known as G protein-coupled estrogen receptor 1 GPER) in human PDL cells and its regulation by IL-1β. IL-1β-induced GPR30 expression in human PDL cells leads to the activation of multiple signaling pathways, including MAPK, NF-κB and PI3K. In contrast, genistein, an estrogen receptor ligand, postpones the activation of MAPKs induced by IL-1β. Moreover, the inhibition of GPR30 by G15, a GPR30-specific antagonist, eliminates this delay. Thus, genistein plays a role in the regulation of MAPK activation via GPR30, and GPR30 represents a novel target regulated by steroid hormones in PDL cells.

  11. Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells.

    PubMed

    Pelaia, Girolamo; Cuda, Giovanni; Vatrella, Alessandro; Gallelli, Luca; Fratto, Donatella; Gioffrè, Vincenza; D'Agostino, Bruno; Caputi, Mario; Maselli, Rosario; Rossi, Francesco; Costanzo, Francesco S; Marsico, Serafino A

    2004-09-01

    The airway epithelium is continuously exposed to inhaled oxidants, including airborne pollutants and cigarette smoke, which can exert harmful proinflammatory and cytotoxic effects. Therefore, the aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the signal transduction pathways activated by increasing concentrations (0.25, 0.5, and 1 mM) of hydrogen peroxide (H(2)O(2)), as well as their effects on IL-8 production and cell viability. The reported results show that H(2)O(2) elicited, in a concentration-dependent fashion, a remarkable increase in phosphorylation-dependent activation of mitogen-activated protein kinases (MAPKs), associated with a significant induction of IL-8 synthesis and a dramatically enhanced cell death. Pre-treatment of HBEC with MAPK inhibitors was able to significantly inhibit the effects of H(2)O(2) on IL-8 secretion, and to effectively prevent cell death. Therefore, these findings suggest that MAPKs play a key role as molecular transducers of the airway epithelial injury triggered by oxidative stress, as well as potential pharmacologic targets for indirect antioxidant intervention.

  12. 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

  13. 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.

  14. Selenium inhibits Staphylococcus aureus-induced inflammation by suppressing the activation of the NF-κB and MAPK signalling pathways in RAW264.7 macrophages.

    PubMed

    Bi, Chong-Liang; Wang, Heng; Wang, Yin-Jie; Sun, Jun; Dong, Jun-Sheng; Meng, Xia; Li, Jian-Ji

    2016-06-05

    Inflammation is the hallmark of Staphylococcus aureus (S. aureus)-induced mastitis. Given the interesting relationship between selenium levels and inflammation, this study aimed to demonstrate that selenium modulated the inflammation reaction by suppressing the nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) signalling pathways. RAW264.7 macrophages were treated with three different concentrations (1μmol/l, 1.5μmol/l, and 2μmol/l) of Na2SeO3 for 12h before infection with S. aureus for 6h, 8h, and 10h. The results showed that selenium significantly reduced the mRNA expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Furthermore, the release of TNF-α, IL-1β, and IL-6 was decreased significantly with selenium supplementation. In addition, selenium influenced the NF-κB signalling pathway by suppressing the activation of NF-κB p65 and degradation of inhibitory kappa-B (IκB). Selenium also suppressed extracellular regulated protein kinase (Erk), c-Jun N-terminal kinase (Jnk), and p38 phosphorylation through the MAPK signalling pathway. In conclusion, selenium played an anti-inflammation role in RAW264.7 macrophages infected with S. aureus by suppressing the activation of the NF-κB and MAPK signalling pathways.

  15. Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways.

    PubMed

    Fernández, Gabriela C; Ilarregui, Juan M; Rubel, Carolina J; Toscano, Marta A; Gómez, Sonia A; Beigier Bompadre, Macarena; Isturiz, Martín A; Rabinovich, Gabriel A; Palermo, Marina S

    2005-05-01

    Galectin-3 (Gal-3), a member of a family of highly conserved carbohydrate-binding proteins, has recently emerged as a novel cellular modulator at inflammatory foci. Here we investigated the effects of Gal-3 on central effector functions of human neutrophils, including phagocytosis, exocytosis of secretory granules, and survival. We examined the effects of Gal-3 alone or in combination with soluble fibrinogen (sFbg), an extracellular mediator that plays a key role during the early phase of the inflammatory response through binding to integrin receptors. In addition we evaluated the intracellular signals triggered by these mediators in human neutrophils. Human neutrophils incubated with recombinant Gal-3 alone increased their phagocytic activity and CD66 surface expression. In contrast to the known antiapoptotic effect of Gal-3 on many cellular types, Gal-3 enhanced PMN apoptotic rate. Preincubation with Gal-3 primed neutrophils to the effects of sFbg, resulting in a synergistic action on degranulation. On the other hand, Gal-3 and sFbg had opposite effects on PMN survival, and the simultaneous action of both agonists partially counteracted the proapoptotic effects of Gal-3. In addition, although sFbg induced its effects through the activation of the ERKs, Gal-3 led to p38 phosphorylation. Disruption of this signaling pathway abrogated Gal-3-mediated modulation of neutrophil degranulation, phagocytosis, and apoptosis. Together, our results support the notion that Gal-3 and sFbg are two physiological mediators present at inflammatory sites that activate different components of the MAPK pathway and could be acting in concert to modulate the functionality and life span of neutrophils.

  16. 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.

  17. 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.

  18. 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.

  19. Consequences of RAS and MAPK activation in the ovary: the good, the bad and the ugly.

    PubMed

    Fan, Heng-Yu; Liu, Zhilin; Mullany, Lisa K; Richards, JoAnne S

    2012-06-05

    This review summarizes studies providing evidence (1) that endogenous RAS activation regulates important physiological events during ovulation and luteinization (2) that expression of the mutant, active KRAS(G12D) in granulosa cells in vivo causes abnormal follicle growth arrest leading to premature ovarian failure and (3) that KRAS(G12D) expression in ovarian surface epithelial (OSE) cells renders them susceptible to the pathological outcome of transformation and tumor formation. These diverse effects of RAS highlight how critical its activation is linked to cell- and stage-specific events in the ovary that control normal processes and that can also lead to altered granulosa cell and OSE cell fates.

  20. The immunostimulating activity of quercetin 3-O-xyloside in murine macrophages via activation of the ASK1/MAPK/NF-κB signaling pathway.

    PubMed

    Lee, Jisun; Choi, Ji Won; Sohng, Jae Kyung; Pandey, Ramesh Prasad; Park, Yong Il

    2016-02-01

    Quercetin is a natural plant flavonoid that has been reported to possess a wide range of beneficial health effects, including anti-cancer and anti-inflammatory activities. Glycosylation of natural flavonoids with various sugar moieties can affect their physical, chemical, and biological properties. In this study, quercetin 3-O-xyloside (Quer-xyl) was enzymatically synthesized, and the immunomodulatory activities of quercetin and Quer-xyl were evaluated and compared. The results showed that Quer-xyl more effectively induced the secretion of TNF-α and IL-6 than quercetin by 2.5 and 1.5-fold, respectively. Quer-xyl dose-dependently induced the inducible nitric oxide synthase (iNOS) expression and increased the production of nitric oxide (NO) 1.3-fold more than quercetin. Quer-xyl also increased the phosphorylation of ASK1 and MAPKs (JNK and p38). Treatment with NQDI-1 (an inhibitor of ASK1) significantly attenuated the Quer-xyl-induced up-regulation of TNF-α secretion. The activation and subsequent nuclear translocation of NF-κB were substantially enhanced upon treatment with Quer-xyl (2.5-20 μM), while NQDI-1 treatment blocked the nuclear translocation of NF-κB. These results demonstrated that Quer-xyl can enhance the early innate immunity more effectively than quercetin by activating macrophages to secrete TNF-α and IL-6 through up-regulation of the redox-dependent ASK1/MAPK/NF-κB signaling pathway, suggesting for the first time that Quer-xyl may represent a new immunostimulator.

  1. 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.

  2. 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

    2016-03-01

    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.

  3. 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

  4. Cytokine Regulation by MAPK Activated Kinase 2 in Keratinocytes Exposed to Sulfur Mustard

    DTIC Science & Technology

    2013-07-10

    per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and...completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information...mRNA levels by destabilizing transcripts via mechanisms that involve exosome recruitment and increased exonuclease activity (Anderson, 2008

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK

    PubMed Central

    Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana

    2017-01-01

    Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935

  10. 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.

  11. A novel brain tumour model in zebrafish reveals the role of YAP activation in MAPK- and PI3K-induced malignant growth

    PubMed Central

    Mayrhofer, Marie; Gourain, Victor; Reischl, Markus; Affaticati, Pierre; Jenett, Arnim; Joly, Jean-Stephane; Benelli, Matteo; Demichelis, Francesca; Poliani, Pietro Luigi; Sieger, Dirk

    2017-01-01

    ABSTRACT Somatic mutations activating MAPK and PI3K signalling play a pivotal role in both tumours and brain developmental disorders. We developed a zebrafish model of brain tumours based on somatic expression of oncogenes that activate MAPK and PI3K signalling in neural progenitor cells and found that HRASV12 was the most effective in inducing both heterotopia and invasive tumours. Tumours, but not heterotopias, require persistent activation of phospho (p)-ERK and express a gene signature similar to the mesenchymal glioblastoma subtype, with a strong YAP component. Application of an eight-gene signature to human brain tumours establishes that YAP activation distinguishes between mesenchymal glioblastoma and low grade glioma in a wide The Cancer Genome Atlas (TCGA) sample set including gliomas and glioblastomas (GBMs). This suggests that the activation of YAP might be an important event in brain tumour development, promoting malignant versus benign brain lesions. Indeed, co-expression of dominant-active YAP (YAPS5A) and HRASV12 abolishes the development of heterotopias and leads to the sole development of aggressive tumours. Thus, we have developed a model proving that neurodevelopmental disorders and brain tumours might originate from the same activation of oncogenes through somatic mutations, and established that YAP activation is a hallmark of malignant brain tumours. PMID:27935819

  12. IGFBP3 and MAPK/ERK signaling mediates melatonin-induced antitumor activity in prostate cancer.

    PubMed

    Mayo, Juan C; Hevia, David; Quiros-Gonzalez, Isabel; Rodriguez-Garcia, Aida; Gonzalez-Menendez, Pedro; Cepas, Vanesa; Gonzalez-Pola, Iván; Sainz, Rosa M

    2017-01-01

    Treatment of prostate cancer (PCa), a leading cause of cancer among males, lacks successful strategies especially in advanced, hormone-refractory stages. Some clinical studies have shown an increase in neuroendocrine-like cells parallel to the tumor progression but their exact role is a matter of debate. The prostate is a well-known target for melatonin, which reduces PCa cells proliferation and induces neuroendocrine differentiation. To evaluate the mechanisms underlying the indole effects on neuroendocrine differentiation and its impact on PCa progression, we used a cell culture model (LNCaP) and a murine model (TRAMP). Persistent ERK1/2 activation was found in both, melatonin and androgen-deprived cells. Melatonin blocked nuclear translocation of androgen receptor (AR), thus confirming anti-androgenic actions of the indole. However, using a comparative genome microarray to check the differentially expressed genes in control, melatonin, or androgen-deprived cells, some differences were found, suggesting a more complex role of the indole. By comparing control cells with those treated with melatonin or depleted of androgen, a cluster of 26 differentially expressed genes (±2.5-fold) was found. Kallikreins (KLK)2 and KLK3 (PSA) were dramatically downregulated by both treatments whereas IGFBP3 and IGF1R were up- and downregulated, respectively, in both experimental groups, thus showing a role for IGF in both scenarios. Finally, melatonin prolonged the survival of TRAMP mice by 33% when given at the beginning or at advances stages of the tumor. Serum IGFBP3 was significantly elevated by the indole in early stages of the tumor, confirming in vivo the role of the IGF signaling in the oncostatic action of the indole.

  13. 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

  14. The Histamine H3 Receptor Differentially Modulates Mitogen-activated Protein Kinase (MAPK) and Akt Signaling in Striatonigral and Striatopallidal Neurons.

    PubMed

    Rapanelli, Maximiliano; Frick, Luciana R; Horn, Kyla D; Schwarcz, Rivka C; Pogorelov, Vladimir; Nairn, Angus C; Pittenger, Christopher

    2016-09-30

    The basal ganglia have a central role in motor patterning, habits, motivated behaviors, and cognition as well as in numerous neuropsychiatric disorders. Receptors for histamine, especially the H3 receptor (H3R), are highly expressed in the striatum, the primary input nucleus of the basal ganglia, but their effects on this circuitry have been little explored. H3R interacts with dopamine (DA) receptors ex vivo; the nature and functional importance of these interactions in vivo remain obscure. We found H3R activation with the agonist R-(-)-α-methylhistamine to produce a unique time- and cell type-dependent profile of molecular signaling events in the striatum. H3 agonist treatment did not detectably alter extracellular DA levels or signaling through the cAMP/DARPP-32 signaling pathway in either D1- or D2-expressing striatal medium spiny neurons (MSNs). In D1-MSNs, H3 agonist treatment transiently activated MAPK signaling and phosphorylation of rpS6 and led to phosphorylation of GSK3β-Ser(9), a novel effect. Consequences of H3 activation in D2-MSNs were completely different. MAPK signaling was unchanged, and GSK3β-Ser(9) phosphorylation was reduced. At the behavioral level, two H3 agonists had no significant effect on locomotion or stereotypy, but they dramatically attenuated the locomotor activation produced by the D1 agonist SKF82958. H3 agonist co-administration blocked the activation of MAPK signaling and the phosphorylation of rpS6 produced by D1 activation in D1-MSNs, paralleling behavioral effects. In contrast, GSK3β-Ser(9) phosphorylation was seen only after H3 agonist treatment, with no interactive effects. H3R signaling has been neglected in models of basal ganglia function and has implications for a range of pathophysiologies.

  15. Involvement of MAPK/NF-κB Signaling in the Activation of the Cholinergic Anti-Inflammatory Pathway in Experimental Colitis by Chronic Vagus Nerve Stimulation

    PubMed Central

    Sun, Peng; Zhou, Kewen; Wang, Sheng; Li, Ping; Chen, Sijuan; Lin, Guiping; Zhao, Yan; Wang, Tinghuai

    2013-01-01

    Background Autonomic nervous system dysfunction is implicated in the etiopathogenesis of inflammatory bowel diseases (IBD). Therapies that increase cardiovagal activity, such as Mind-Body interventions, are currently confirmed to be effective in clinical trials in IBD. However, a poor understanding of pathophysiological mechanisms limits the popularization of therapies in clinical practice. The aim of the present study was to explore the mechanisms of these therapies against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats using a chronic vagus nerve stimulation model in vivo, as well as the lipopolysaccharide (LPS)-induced inflammatory response in human epithelial colorectal adenocarcinoma cells (Caco-2) by acetylcholine in vitro. Methods and Results Colitis was induced in rats with rectal instillation of TNBS, and the effect of chronic VNS (0.25 mA, 20 Hz, 500 ms) on colonic inflammation was evaluated. Inflammatory responses were assessed by disease activity index (DAI), histological scores, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS), TNF-α and IL-6 production. The expression of Mitogen-activated protein kinases (MAPK) family members, IκB-α, and nuclear NF-κB p65 were studied by immunoblotting. Heart rate variability (HRV) analysis was also applied to assess the sympathetic-vagal balance. DAI, histological scores, MPO activity, iNOS, TNF-α and IL-6 levels were significantly decreased by chronic VNS. Moreover, both VNS and acetylcholine reduced the phosphorylation of MAPKs and prevented the nuclear translocation of NF-κB p65. Methyllycaconitine (MLA) only reversed the inhibitory effect on p-ERK and intranuclear NF-κB p65 expression by ACh in vitro, no significant change was observed in the expression of p-p38 MAPK or p-JNK by MLA. Conclusion Vagal activity modification contributes to the beneficial effects of the cholinergic anti-inflammatory pathway in IBD-related inflamed colonic mucosa based on the activation

  16. Microcystin-LR exhibits immunomodulatory role in mouse primary hepatocytes through activation of the NF-κB and MAPK signaling pathways.

    PubMed

    Zhang, Jianying; Chen, Jin; Xia, Zongping

    2013-11-01

    Microcystin-leucine-arginine (MCLR) is an environmental toxin from harmful algae, which has been linked to hepatotoxicity with high risks associated with liver disease. In this study, we explored the role of MCLR in NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, which are important in regulating inflammatory and immune responses, in human hepatoma cell line HepG2 and primary mouse hepatocytes (PMHs). By in vitro cell-free and luciferase reporter systems, Western blotting with antiphospho-inhibitory protein of NF-κB (IκBα)/c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase 1/2 (ERK1/2) antibody, it was found that at noncytotoxic concentrations (≤ 20 nM MCLR in PMHs, 1-1000 nM in HepG2), MCLR treatment alone promoted activation of NF-κB and MAPK pathways and modulated TNF-α-induced activation of the 2 pathways in both cell models. By ELISA assay, MCLR was found to induce production of proinflammatory cytokine IL-6 in PMHs. At cytotoxic concentrations (≥ 50 nM MCLR in PMHs), MCLR dramatically reduced cell viability and damaged cell morphology in PMHs, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and transmission electron microscopy analysis. These results suggest that MCLR below 20 nM has significant immunomodulatory activities through activation of NF-κB and MAPK signaling pathways, and PMHs are more sensitive to MCLR-induced cytotoxicity than HepG2. To our knowledge, this is the first report showing the immunomodulatory role of MCLR in hepatocytes. Our results provide a better understanding of the molecular mechanisms underlying MCLR-induced hepatotoxicity.

  17. (+)-Catechin Attenuates NF-κB Activation Through Regulation of Akt, MAPK, and AMPK Signaling Pathways in LPS-Induced BV-2 Microglial Cells.

    PubMed

    Syed Hussein, Sharifah Salwa; Kamarudin, Muhamad Noor Alfarizal; Kadir, Habsah Abdul

    2015-01-01

    (+)-Catechin is a flavanol that possesses various health and medicinal values, which include neuroprotection, anti-oxidation, antitumor and antihepatitis activities. This study investigated the modulatory effects of (+)-catechin on the lipopolysaccharides (LPS)-stimulated BV-2 cells. (+)-catechin attenuated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and inhibited microglial NO and ROS production. Additionally, (+)-catechin suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, while augmenting IL-4. (+)-catechin attenuated LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation via the inhibition of IκB-α phosphorylation. Moreover, (+)-catechin blocked the activation of Akt and its inhibition was shown to play a crucial role in LPS-induced inflammation in BV-2 microglial cells. (+)-catechin also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), and p-38 mitogen activated protein kinases (p38 MAPK) and specific inhibitors of ERK1/2 (UO126) and p38 MAPK (SB202190) subsequently down-regulated the expression of the proinflammatory mediators iNOS and COX-2. Further mechanistic study revealed that (+)-catechin acted through the amelioration of the LPS-induced suppression of adenosine monophosphate-activated protein kinase (AMPK) activity. Taken together, our data indicate that (+)-catechin exhibits anti-inflammatory effects in BV-2 cells by suppressing the production of proinflammatory mediators and mitigation of NF-κB through Akt, ERK, p38 MAPK, and AMPK pathways.

  18. The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research.

    PubMed

    Menon, Manoj B; Dhamija, Sonam; Kotlyarov, Alexey; Gaestel, Matthias

    2015-01-01

    In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/β inhibitors. In this "Letter to the editor" we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.

  19. Costunolide inhibits interleukin-1beta expression by down-regulation of AP-1 and MAPK activity in LPS-stimulated RAW 264.7 cells.

    PubMed

    Kang, Jong Soon; Yoon, Yeo Dae; Lee, Ki Hoon; Park, Song-Kyu; Kim, Hwan Mook

    2004-01-02

    Costunolide, a sesquiterpene lactone isolated from the root of Saussurea lappa Clarke, is known to have a variety of biological activities, including anti-carcinogenic and anti-fungal activities. Here, we demonstrated the inhibitory effect of costunolide on the protein and mRNA expression of interleukin-1beta (IL-1beta) in LPS-stimulated RAW 264.7 cells. We also showed that costunolide suppressed the transcriptional activity of the IL-1beta promoter. Moreover, costunolide inhibited the activity of AP-1 transcription factor, and the phosphorylation of MAPKs, including SAPK/JNK and p38 MAP kinase. The inhibitory effect of costunolide on AP-1 activity was also confirmed by an electrophoretic mobility shift assay. Additionally, specific inhibitors of SAPK/JNK and p38 MAP kinase, SP600125 and SB203580, also suppressed LPS-induced increase in IL-1beta gene expression and AP-1 DNA binding. Taken together, these results demonstrate that costunolide inhibits IL-1beta gene expression by blocking the activation of MAPKs and DNA binding of AP-1 in LPS-stimulated RAW 264.7 cells.

  20. Rationally designed treatment for solid tumors with MAPK pathway activation: a Phase I study of paclitaxel and bortezomib using an adaptive dose-finding approach

    PubMed Central

    Mehnert, Janice M.; Tan, Antoinette R.; Moss, Rebecca; Poplin, Elizabeth; Stein, Mark N.; Sovak, Mika; Levinson, Kelly; Lin, Hongxia; Kane, Michael; Gounder, Murugesan; Lin, Yong; Shih, Weichung Joe; White, Eileen; Rubin, Eric H.; Karantza, Vassiliki

    2011-01-01

    In the preclinical setting, phosphorylation and subsequent proteosomal degradation of the proapoptotic protein BIM confers resistance to paclitaxel in solid tumors with RAS/RAF/MAPK pathway activation. Concurrent administration of the proteasome inhibitor bortezomib enables paclitaxel-induced BIM accumulation, restoring cancer cell apoptosis in vitro and producing tumor regression in mice in vivo. A Phase I study was conducted to determine the MTD of paclitaxel and bortezomib combinatorial treatment. Sixteen patients with refractory solid tumors commonly exhibiting MAPK pathway activation were treated with weekly paclitaxel and bortezomib. Starting doses were 40 mg/m2 for paclitaxel and 0.7 mg/m2 for bortezomib. A modified continual reassessment method (MCRM) adapted for 2-drug escalation was used for MTD determination with 3-patient cohorts treated at each dose level. MTD was reached at 60 mg/m2 paclitaxel and 1.0 mg/m2 bortezomib, the recommended phase II dose. Therapy was overall well tolerated. Most frequently observed toxicities included anemia (in 43.75% of patients, one Grade 3 event), fatigue (in 43.75% of patients, one Grade 3 event beyond cycle 1) and neuropathy (in 31.25% of patients, one Grade 3 event after cycle 1). Of 15 evaluable patients, one NSCLC patient with paclitaxel exposure at the adjuvant setting had a PR and five patients had SD; median disease stabilization was 143.5 days; three NSCLC patients had SD lasting 165 days or longer. Thus, rationally designed weekly treatment with paclitaxel and bortezomib in solid tumors with MAPK pathway activation, including previously taxane-treated malignancies, is a tolerable regimen with preliminary signals of antitumor activity worthy of further investigation. PMID:21680752

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Effect of hyperprolactinemia on PRL-receptor expression and activation of Stat and Mapk cell signaling in the prostate of long-term sexually-active rats.

    PubMed

    Pascual-Mathey, Luz I; Rojas-Duran, Fausto; Aranda-Abreu, Gonzalo E; Manzo, Jorge; Herrera-Covarrubias, Deissy; Muñoz-Zavaleta, David A; Garcia, Luis I; Hernandez, Ma Elena

    2016-04-01

    The abnormal elevation of serum PRL, referred to as hyperprolactinemia (HyperPRL), produces alterations in several reproductive parameters of male rats such as penile erection or decreased tendency to reach ejaculation. Additionally, this situation produces a significant modification of prostate histology, as observed in the epithelial structure and alveolar area, which could reach a level of hyperplasia in the long-term. In this tissue, HyperPRL produces an increase in expression of PRL receptors and activation of the Stat3 signaling pathway that is correlated with the evolution of prostate pathologies. However, the impact of HyperPRL in long-term sexually active male rats is unknown. In this work, using constantly copulating Wistar male rats with induced HyperPRL, we analyzed the level of serum PRL, the effect on prostate PRL receptors, and activation of pStat3, pStat5 and Mapk signaling pathways. Two procedures to induce HyperPRL were employed, comprising daily IP administration or adenohypophysis transplant, and although neither affected the execution of sexual behavior, the serum PRL profile following successive ejaculations was affected. Messenger RNA expression of the short and long isoforms of the PRL receptor at the ventral prostate was affected in different ways depending on the procedure to induce HyperPRL. The ventral prostate did not show any modification in terms of activation of the pStat5 signaling pathway in subjects with daily administration of PRL, although this was significantly increased in ADH transplanted subjects in the second and fourth consecutive ejaculation. A similar profile was found for the pStat3 pathway which additionally showed a significant increase in the third and fourth ejaculation of daily-injected subjects. The Mapk signaling pathway did not show any modifications in subjects with daily administration of PRL, but showed a significant increase in the second and third ejaculations of subjects with ADH transplants. Thus

  6. Chloroacetic acid induced neuronal cells death through oxidative stress-mediated p38-MAPK activation pathway regulated mitochondria-dependent apoptotic signals.

    PubMed

    Chen, Chun-Hung; Chen, Sz-Jie; Su, Chin-Chuan; Yen, Cheng-Chieh; Tseng, To-Jung; Jinn, Tzyy-Rong; Tang, Feng-Cheng; Chen, Kuo-Liang; Su, Yi-Chang; Lee, kuan-I; Hung, Dong-Zong; Huang, Chun-Fa

    2013-01-07

    Chloroacetic acid (CA), a toxic chlorinated analog of acetic acid, is widely used in chemical industries as an herbicide, detergent, and disinfectant, and chemical intermediates that are formed during the synthesis of various products. In addition, CA has been found as a by-product of chlorination disinfection of drinking water. However, there is little known about neurotoxic injuries of CA on the mammalian, the toxic effects and molecular mechanisms of CA-induced neuronal cell injury are mostly unknown. In this study, we examined the cytotoxicity of CA on cultured Neuro-2a cells and investigated the possible mechanisms of CA-induced neurotoxicity. Treatment of Neuro-2a cells with CA significantly reduced the number of viable cells (in a dose-dependent manner with a range from 0.1 to 3mM), increased the generation of ROS, and reduced the intracellular levels of glutathione depletion. CA also increased the number of sub-G1 hypodiploid cells; increased mitochondrial dysfunction (loss of MMP, cytochrome c release, and accompanied by Bcl-2 and Mcl-1 down-regulation and Bax up-regulation), and activated the caspase cascades activations, which displayed features of mitochondria-dependent apoptosis pathway. These CA-induced apoptosis-related signals were markedly prevented by the antioxidant N-acetylcysteine (NAC). Moreover, CA activated the JNK and p38-MAPK pathways, but did not that ERK1/2 pathway, in treated Neuro-2a cells. Pretreatment with NAC and specific p38-MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125) effectively abrogated the phosphorylation of p38-MAPK and attenuated the apoptotic signals (including: decrease in cytotoxicity, caspase-3/-7 activation, the cytosolic cytochrome c release, and the reversed alteration of Bcl-2 and Bax mRNA) in CA-treated Neuro-2a cells. Taken together, these data suggest that oxidative stress-induced p38-MAPK activated pathway-regulated mitochondria-dependent apoptosis plays an important role in CA-caused neuronal cell

  7. 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

    2015-10-05

    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.

  8. Caenorhabditis elegans mom-4 is required for the activation of the p38 MAPK signaling pathway in the response to Pseudomonas aeruginosa infection.

    PubMed

    Xu, Ajing; Shi, Guojun; Liu, Feng; Ge, Baoxue

    2013-01-01

    The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. Aeruginosa.

  9. 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

    2016-06-08

    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.

  10. 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

  11. Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells.

    PubMed

    Park, Moon-Taek; Choi, Jung-A; Kim, Min-Jeong; Um, Hong-Duck; Bae, Sangwoo; Kang, Chang-Mo; Cho, Chul-Koo; Kang, Seongman; Chung, Hee Yong; Lee, Yun-Sil; Lee, Su-Jae

    2003-12-12

    We previously demonstrated that the phytosphingosine-induced apoptosis was accompanied by the concomitant induction of both the caspase-8-mediated and mitochondrial activation-mediated apoptosis pathways. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in the activation of these two distinct cell death pathways induced by phytosphingosine in human cancer cells. Phytosphingosine caused strong induction of caspase-8 activity and caspase-independent Bax translocation to the mitochondria. A rapid decrease of phosphorylated ERK1/2 and a marked increase of p38 MAPK phosphorylation were observed within 10 min after phytosphingosine treatment. Activation of ERK1/2 by pretreatment with phorbol 12-myristate 13-acetate or forced expression of ERK1/2 attenuated phytosphingosine-induced caspase-8 activation. However, Bax translocation and caspase-9 activation was unaffected, indicating that down-regulation of the ERK activity is specifically required for the phytosphingosine-induced caspase-8-dependent cell death pathway. On the other hand, treatment with SB203580, a p38 MAPK-specific inhibitor, or expression of a dominant negative form of p38 MAPK suppressed phytosphingosine-induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation but did not affect caspase-8 activation, indicating that activation of p38 MAPK is involved in the mitochondrial activation-mediated cell death pathway. Our results suggest that phytosphingosine can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, enhancing the understanding of the molecular mechanisms utilized by naturally occurring metabolites to regulate cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both our understanding of cell death events and development of cancer therapeutic agents.

  12. Neuroprotection by Imipramine against lipopolysaccharide-induced apoptosis in hippocampus-derived neural stem cells mediated by activation of BDNF and the MAPK pathway.

    PubMed

    Peng, Chi-Hsien; Chiou, Shih-Hwa; Chen, Shih-Jen; Chou, Yueh-Ching; Ku, Hung-Hai; Cheng, Cheng-Kuo; Yen, Chih-Ju; Tsai, Tung-Hu; Chang, Yuh-Lih; Kao, Chun-Lan

    2008-02-01

    Depression is accompanied by the activation of the inflammatory-response system, and increased production of proinflammatory cytokines may play a role in the pathophysiology of depressive disorders. Imipramine (IM), a tricyclic antidepressant drug, has recently been shown to promote neurogenesis and improve the survival rate of neurons in the hippocampus. However, whether IM elicits a neuroprotective or anti-inflammatory effect, or promotes the differentiation of neural stem cells (NSCs) remains to be elucidated. In this study, we cultured NSCs derived from the hippocampal tissues of adult rats as an in vitro model to evaluate the NSCs drug-modulation effects of IM. Our results showed that 3 microM IM treatment significantly increased the survival rate of NSCs, and up-regulated the mRNA and protein expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 in Day-7 IM-treated NSCs. Similar to BDNF-treated effect, incubation of NSCs with 3 microM IM increased Bcl-2 protein levels and further prevented lipopolysaccharide (LPS)-induced apoptosis through the activation of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway. Inhibition of BDNF expression with small interfering RNA (siRNA), or blocking the MAPK pathway with U0126 further significantly decreased Bcl-2 protein levels and abrogated the neuroprotective effects of IM against LPS-induced apoptosis in NSCs. In addition, the percentages of serotonin and MAP-2-positive neuronal cells in the Day 7 culture of IM-treated NSCs were significantly increased. By using microdialysis with high performance liquid chromatography-electrochemical detection, the functional release of serotonin in the process of serotoninergic differentiation of IM-treated NSCs was concomitantly increasing and mediated by the activation of the BDNF/MAPK/ERK pathway/Bcl-2 cascades. In sum, the study results indicate that IM can increase the neuroprotective effects, suppress the LPS-induced inflammatory

  13. Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1/2 MAPKs.

    PubMed

    Lee, H-Y; Kim, J; Noh, H J; Kim, H-P; Park, S-J

    2014-12-01

    Much remains unknown about the mammalian immune response to Giardia lamblia, a protozoan pathogen that causes diarrhoeal outbreaks. We fractionated protein extracts of G. lamblia trophozoites by Viva-spin centrifugation, DEAE ion exchange and gel filtration chromatography. Resultant fractions were screened for antigenic molecules by western blots analysis using anti-G. lamblia antibodies (Abs), resulting in identification of G. lamblia binding immunoglobulin protein (GlBiP). Maturation of mouse dendritic cells (DCs) in response to recombinant GlBiP (rGlBiP) was detected by increased expression of surface molecules such as CD80, CD86 and MHC class II; these mature DCs, produced pro-inflammatory cytokines (TNF-α, IL-12 and IL-6). Especially, the truncated rGlBiP containing the heat-shock protein 70 domain-induced cytokine production from mouse DCs. rGlBiP-induced DC activation was initiated by TLR4 in a MyD88-dependent way and occurred through activation of p38 and ERK1/2 MAPKs as well as increased activity of NF-κB and AP-1. Moreover, CD4(+) T cells stimulated with rGlBiP-treated DCs produced high levels of IL-2 and IFN-γ. Together, our results suggest that GlBiP contributes to maturation of DCs via activation of TLR4-MyD88-p38, ERK1/2 MAPK, NF-κB and AP-1.

  14. 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.

  15. 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.

  16. 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

  17. Novel Noncatalytic Substrate-Selective p38α-Specific MAPK Inhibitors with Endothelial-Stabilizing and Anti-Inflammatory Activity.

    PubMed

    Shah, Nirav G; Tulapurkar, Mohan E; Ramarathnam, Aparna; Brophy, Amanda; Martinez, Ramon; Hom, Kellie; Hodges, Theresa; Samadani, Ramin; Singh, Ishwar S; MacKerell, Alexander D; Shapiro, Paul; Hasday, Jeffrey D

    2017-04-15

    The p38 MAPK family is composed of four kinases of which p38α/MAPK14 is the major proinflammatory member. These kinases contribute to many inflammatory diseases, but the currently available p38 catalytic inhibitors (e.g., SB203580) are poorly effective and cause toxicity. We reasoned that the failure of catalytic p38 inhibitors may derive from their activity against noninflammatory p38 isoforms (e.g., p38β/MAPK11) and loss of all p38α-dependent responses, including anti-inflammatory, counterregulatory responses via mitogen- and stress-activated kinase (MSK) 1/2 and Smad3. We used computer-aided drug design to target small molecules to a pocket near the p38α glutamate-aspartate (ED) substrate-docking site rather than the catalytic site, the sequence of which had only modest homology among p38 isoforms. We identified a lead compound, UM101, that was at least as effective as SB203580 in stabilizing endothelial barrier function, reducing inflammation, and mitigating LPS-induced mouse lung injury. Differential scanning fluorimetry and saturation transfer difference-nuclear magnetic resonance demonstrated specific binding of UM101 to the computer-aided drug design-targeted pockets in p38α but not p38β. RNA sequencing analysis of TNF-α-stimulated gene expression revealed that UM101 inhibited only 28 of 61 SB203580-inhibited genes and 7 of 15 SB203580-inhibited transcription factors, but spared the anti-inflammatory MSK1/2 pathway. We provide proof of principle that small molecules that target the ED substrate-docking site may exert anti-inflammatory effects similar to the catalytic p38 inhibitors, but their isoform specificity and substrate selectivity may confer inherent advantages over catalytic inhibitors for treating inflammatory diseases.

  18. Lycium barbarum polysaccharide LBPF4-OL may be a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer.

    PubMed

    Zhang, Xiao-rui; Qi, Chun-hui; Cheng, Jun-ping; Liu, Gang; Huang, Lin-juan; Wang, Zhong-fu; Zhou, Wen-xia; Zhang, Yong-xiang

    2014-03-01

    Recognition of the utility of the traditional Chinese medicine Lycium barbarum L. has been gradually increasing in Europe and the Americas. Many immunoregulation and antitumor effects of L. barbarum polysaccharides (LBP) have been reported, but its molecular mechanism is not yet clear. In this study, we reported that the activity of the polysaccharide LBPF4-OL, which was purified from LBP, is closely associated with the TLR4-MAPK signaling pathway. We found that LBPF4-OL can significantly induce TNF-α and IL-1β production in peritoneal macrophages isolated from wild-type (C3H/HeN) but not TLR4-deficient mice (C3H/HeJ). We also determined that the proliferation of LBPF4-OL-stimulated lymphocytes from C3H/HeJ mice is significantly weaker than that of lymphocytes from C3H/HeN mice. Furthermore, through a bio-layer interferometry assay, we found that LPS but not LBPF4-OL can directly associate with the TLR4/MD2 molecular complex. Flow cytometry analysis indicated that LBPF4-OL markedly upregulates TLR4/MD2 expression in both peritoneal macrophages and Raw264.7 cells. As its mechanism of action, LBPF4-OL increases the phosphorylation of p38-MAPK and inhibits the phosphorylation of JNK and ERK1/2, as was observed through Western blot analysis. These data suggest that the L. barbarum polysaccharide LBPF4-OL is a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer.

  19. 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

  20. Src kinase integrates PI3K/Akt and MAPK/ERK1/2 pathways in T3-induced Na-K-ATPase activity in adult rat alveolar cells.

    PubMed

    Lei, Jianxun; Ingbar, David H

    2011-11-01

    We previously reported that the 3,5,3'-triiodo-L-thyronine (T3)-induced increase of Na-K-ATPase activity in rat alveolar epithelial cells (AECs) required activation of Src kinase, PI3K, and MAPK/ERK1/2. In the present study, we assessed the role of Akt in Na-K-ATPase activity and the interaction between the PI3K and MAPK in response to T3 by using MP48 cells, inhibitors, and constitutively active mutants in the MP48 (alveolar type II-like) cell line. The Akt inhibitor VIII blocked T3-induced increases in Na-K-ATPase activity and amount of plasma membrane Na-K-ATPase protein. The Akt inhibitor VIII also abolished the increase in Na-K-ATPase activity induced by constitutively active mutants of either Src kinase or PI3K. Moreover, constitutively active mutants of Akt increased Na-K-ATPase activity in the absence of T3. Thus activation of Akt was required for T3-induced Na-K-ATPase activity in AECs and is sufficient in the absence of T3. Inhibitors of Src kinase (PP1), PI3K (wortmannin), and ERK1/2 (U0126) all blocked the T3-induced Na-K-ATPase activity. PP1 blocked the activation of PI3K and also ERK1/2 by T3, whereas U0126 did not prevent T3 activation of Src kinase or PI3K activity. Wortmannin did not significantly alter T3-increased MAPK/ERK1/2 activity, suggesting that T3-activated PI3K/Akt and MAPK/ERK1/2 pathways acted downstream of the Src kinase. Furthermore, in the absence of T3, a constitutively active mutant of Src kinase increased activities of Na-K-ATPase, PI3K, and MAPK/ERK1/2. A constitutively active mutant of PI3K enhanced Na-K-ATPase activity but did not alter the MAPK/ERK1/2 activity significantly. In summary, in adult rat AECs T3-stimulated Src kinase activity can activate both PI3K/Akt and MAPK/ERK1/2, and activation of Akt is necessary for T3-induced Na-K-ATPase activity.

  1. PRDX1 is involved in palmitate induced insulin resistance via regulating the activity of p38MAPK in HepG2 cells.

    PubMed

    Tang, Zhuqi; Xia, Nana; Yuan, Xinlu; Zhu, Xiaohui; Xu, Guangfei; Cui, Shiwei; Zhang, Tingting; Zhang, Wanlu; Zhao, Yun; Wang, Suxin; Shi, Bimin

    2015-10-02

    Studies have identified that type 2 diabetes mellitus (T2DM) patients displayed higher levels of plasma peroxiredoxin1(PRDX1) than non-diabetics. However, the impact of PRDX1 on insulin resistance and the underlying mechanism remains totally unknown. Here, we investigated the influence of PRDX1 on hepatic insulin resistance. We showed that the protein and mRNA levels of PRDX1 were significantly elevated under insulin-resistant conditions. In addition, we showed that interference of PRDX1 ameliorated palmitate-induced insulin resistance in HepG2 cells, which was indicated by elevated phosphorylation of protein kinase B (AKT) and of glycogen synthase kinase-3 (GSK3β). Furthermore, the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were down-regulated following PRDX1 depletion. Accordingly, glucose uptake was suppressed in PRDX1-interferred HepG2 cells. In addition, Over-expression of PRDX1 enhanced PA-induced insulin resistance in HepG2 cells. Moreover, we found that knocking down PRDX1 improves insulin sensitivity and decreased the activation of p38 mitogen-activated protein kinase (p38MAPK). Our results demonstrate that PRDX1 can induce hepatic insulin resistance by activating p38MAPK signaling and identifies potential targets for new treatments.

  2. GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) UPREGULATES β1 INTEGRIN AND INCREASES MIGRATION OF HUMAN TROPHOBLAST SWAN 71 CELLS VIA PI3K AND MAPK ACTIVATION

    PubMed Central

    Furmento, Verónica A.; Marino, Julieta; Blank, Viviana C.; Cayrol, María Florencia; Cremaschi, Graciela A.; Aguilar, Rubén C.; Roguin, Leonor P.

    2017-01-01

    Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of β1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of β1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates β1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development. PMID:26992288

  3. Granulocyte colony-stimulating factor (G-CSF) upregulates β1 integrin and increases migration of human trophoblast Swan 71 cells via PI3K and MAPK activation.

    PubMed

    Furmento, Verónica A; Marino, Julieta; Blank, Viviana C; Cayrol, María Florencia; Cremaschi, Graciela A; Aguilar, Rubén C; Roguin, Leonor P

    2016-03-15

    Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of β1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of β1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates β1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development.

  4. 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.

  5. Rga4 modulates the activity of the fission yeast cell integrity MAPK pathway by acting as a Rho2 GTPase-activating protein.

    PubMed

    Soto, Teresa; Villar-Tajadura, Maria Antonia; Madrid, Marisa; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2010-04-09

    Rho GTPase-activating proteins (GAPs) are responsible for the inactivation of Rho GTPases, which are involved in the regulation of critical biological responses in eukaryotic cells, ranging from cell cycle control to cellular morphogenesis. The genome of fission yeast Schizosaccharomyces pombe contains six genes coding for putative Rho GTPases, whereas nine genes code for predicted Rho GAPs (Rga1 to Rga9). One of them, Rga4, has been recently described as a Cdc42 GAP, involved in the control of cell diameter and symmetry in fission yeast. In this work we show that Rga4 is also a Rho2 GAP that negatively modulates the activity of the cell integrity pathway and its main effector, MAPK Pmk1. The DYRK-type protein kinase Pom1, which regulates both the localization and phosphorylation state of Rga4, is also a negative regulator of the Pmk1 pathway, but this control is not dependent upon the Rga4 role as a Rho2-GAP. Hence, two subsets of Rga4 negatively regulate Cdc42 and Rho2 functions in a specific and unrelated way. Finally, we show that Rga7, another Rho2 GAP, down-regulates the Pmk1 pathway in addition to Rga4. These results reinforce the notion of the existence of complex mechanisms determining the selectivity of Rho GAPs toward Rho GTPases and their functions.

  6. 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

  7. Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK.

    PubMed

    Castrillo, A; de Las Heras, B; Hortelano, S; Rodriguez, B; Villar, A; Bosca, L

    2001-05-11

    The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.

  8. Antitumor properties of salinomycin on cisplatin-resistant human ovarian cancer cells in vitro and in vivo: involvement of p38 MAPK activation.

    PubMed

    Zhang, Bei; Wang, Xueya; Cai, Fengfeng; Chen, Weijie; Loesch, Uli; Zhong, Xiao Yan

    2013-04-01

    In order to search for alternative agents to overcome chemoresistance during the treatment of ovarian cancer, this study aimed to examine the anticancer effects and action mechanism of salinomycin, a selective inhibitor of cancer stem cells, on cisplatin-resistant human ovarian cancer cell lines in vitro and in vivo. The concentration- (0.01-200 µM) and time‑dependent (24-72 h) growth inhibitory effects of salinomycin were observed in the ovarian cancer cell lines OV2008, C13, A2780, A2780-cp, SKOV3 and OVCAR3, by measuring cell viability using the resazurin reduction assay. The IC50 (24 h) range of salinomycin on the six cell lines was found to be 1.7-7.4 µM. After cisplatin-resistant C13 cells were treated with salinomycin, the percentage of apoptotic cells determined by flow cytometry was significantly increased, in a concentration- and time‑dependent manner. However, no cell cycle arrest was detected in the G1/G0, S and G2/M phases in the salinomycin‑treated and control cells. The Bio-Plex phosphoprotein 5-plex assay (Akt, IκB-α, ERK1/2, JNK and p38 MAPK) demonstrated a marked time- and concentration‑dependent increase in the phosphorylation of p38 MAPK, subsequent to salinomycin treatment. Moreover, salinomycin significantly suppressed tumor growth in a tumor xenograft model. These findings suggested that salinomycin efficiently inhibits the cisplatin-resistant human ovarian cancer cell line growth through the induction of apoptosis, potentially associated with the p38 MAPK activation.

  9. MAPK Assays in Arabidopsis MAMP-PRR Signal Transduction.

    PubMed

    Chung, Hoo Sun; Sheen, Jen

    2017-01-01

    Activation of MAPK (Mitogen-Activated Protein Kinase) cascades after MAMP (Microbe-Associated Molecular Pattern) perception through PRR (Pattern Recognition Receptor) is one of the first conserved responses when plants encounter microbial organisms. Phosphorylation of various cellular factors in the MAMP-PRR pathway by MAPK cascades is critical for broad-spectrum plant innate immunity. Measurement of MAPK activation and identification of MAPK phosphorylation targets in the MAMP-PRR signal transduction pathway are essential to understand how plants reprogram their cellular processes to cope with unfavorable microbial attack. Here, we describe detailed protocols of three assays measuring MAPK activity after MAMP perception: (1) immune-blotting analysis with anti-phospho ERK1/2 antibody; (2) in-gel kinase assay using a general substrate myelin basic protein (MBP); (3) an in vitro kinase assay to evaluate phosphorylation of MAPK substrate candidates during MAMP-PRR signaling based on a protoplast expression system.

  10. 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.

  11. Mitogen-activated protein kinase (MAPK)-regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program.

    PubMed

    Artigas, Natalia; Ureña, Carlos; Rodríguez-Carballo, Edgardo; Rosa, José Luis; Ventura, Francesc

    2014-09-26

    The transcription factors Runx2 and Osx (Osterix) are required for osteoblast differentiation and bone formation. Runx2 expression occurs at early stages of osteochondroprogenitor determination, followed by Osx induction during osteoblast maturation. We demonstrate that coexpression of Osx and Runx2 leads to cooperative induction of expression of the osteogenic genes Col1a1, Fmod, and Ibsp. Functional interaction of Osx and Runx2 in the regulation of these promoters is mediated by enhancer regions with adjacent Sp1 and Runx2 DNA-binding sites. These enhancers allow formation of a cooperative transcriptional complex, mediated by the binding of Osx and Runx2 to their specific DNA promoter sequences and by the protein-protein interactions between them. We also identified the domains involved in the interaction between Osx and Runx2. These regions contain the amino acids in Osx and Runx2 known to be phosphorylated by p38 and ERK MAPKs. Inhibition of p38 and ERK kinase activities or mutation of their known phosphorylation sites in Osx or Runx2 strongly disrupts their physical interaction and cooperative transcriptional effects. Altogether, our results provide a molecular description of a mechanism for Osx and Runx2 transcriptional cooperation that is subject to further regulation by MAPK-activating signals during osteogenesis.

  12. 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

  13. Lantana macrophylla Schauer (Verbenaceae) ethanolic extract induces activation of ERK1/2 and p38 MAPKs pathway and Ca2+ imbalance in human trophoblasts derived cell lines.

    PubMed

    da Conceição, Aline O; de Oliveira, Fernando F; de Oliveira, Rosilene A; de J da S Junior, Ademir; Takser, Larissa; Reyes-Moreno, Carlos; Lafond, Julie

    2012-03-01

    Lantana macrophylla Schauer (Verbenaceae) a medicinal plant used to treat menstrual and respiratory disorders was investigated. The ethanolic extract from leaves was subjected to phytochemical and biological analysis. BeWo and JEG-3 cells were used to evaluate human chorionic gonadotropin hormone (hCG) production, syncytial formation, Ca2+ uptake and Ca2+ handling protein expression. The cAMP production and the mitogen-activated protein kinases (MAPKs) phosphorylation were also investigated. Phytochemical analysis yield three triterpenes: oleanolic, ursolic and latonolic acid. Viability assay showed no significant cytotoxic effect. A significant decrease in hCG production but not a disturbance on BeWo cell fusion were observed. The cAMP pathway was not affected by L. macrophylla extract alone; although the cAMP production inducted by forskolin was diminished. Both ERK1/2 and p38 MAPKs pathways were activated. Increased intracellular Ca2+ concentration ([Ca2+]i) was observed after 24 h treatment in a time and dose dependent manner; however only L. macrophylla at 10 μg/mL induced increased [Ca2+]i after 10 min treatment. CaBP28K and PMCA1/4 were modulated at protein and mRNA levels, respectively. This study showed for the first time the effect of triterpenoids from L. macrophylla leaves on trophoblasts-like cells and indicates a potential toxic effect of this plant in the placental development and fetal growth.

  14. Pneumolysin induces cellular senescence by increasing ROS production and activation of MAPK/NF-κB signal pathway in glial cells.

    PubMed

    Kwon, Ii-Seul; Kim, Jinwook; Rhee, Dong-Kwon; Kim, Byung-Oh; Pyo, Suhkneung

    2017-02-20

    Senescence is an irreversible proliferation arrest that is induced by various stress stimuli including genotoxin. Pneumolysin (PLY) is a pathogenicity factor unique to Streptococcus pneumoniae that is important in pneumococcal-induced diseases such as otitis media, meningitis and pneumonia. However, the cell fate response to the toxin is mechanistically unclear. We investigated the effect of PLY on cellular senescence in BV-2 microglial cells. Exposure to PLY resulted in changes in the expression of phospho-p53, p21, p16, pRb and CDK2 and increased the number of senescence associated β-gal positive cells. PLY-treatment also increased PAI-1 expression and cell proliferation arrest in concentration- and time-dependent manners. PLY induced NF-κB activation and phosphorylation of SIRT-1, ERK1/2, JNK, and p38 MAPK. In addition, PLY increased the production of reactive oxygen species. Overall, the results suggest that PLY regulates microglial cellular senescence by enhancing production of reactive oxygen species, activation of MAPK and NF-κB, and phosphorylation of SIRT-1.

  15. The disintegrin, trimucrin, suppresses LPS-induced activation of phagocytes primarily through blockade of NF-κB and MAPK activation.

    PubMed

    Hung, Yu-Chun; Hsu, Chun-Chieh; Chung, Ching-Hu; Huang, Tur-Fu

    2016-07-01

    In addition to antiplatelet activity, disintegrin, a small-mass RGD-containing polypeptide, has been shown to exert anti-inflammatory effects but the mechanism involved remains unclear. In this study, we report that trimucrin, a disintegrin from the venom of Trimeresurus mucrosquamatus, inhibits lipopolysaccharide (LPS)-induced stimulation of THP-1 and RAW 264.7 cells. We also investigate the underlying mechanism. Trimucrin decreased the release of proinflammatory cytokines including tumor necrosis factor α (TNFα), interleukin-6 (IL-6), nitric oxide, and reactive oxygen species (ROS), and inhibited the adhesion and migration of LPS-activated phagocytes. Trimucrin significantly blocked the expression of nuclear factor kappaB (NF-κB)-related downstream inducible enzymes such as inducible nitric oxide synthase (iNOS) and COX-2. In addition, its anti-inflammatory effect was associated with the decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, trimucrin concentration dependently inhibited LPS-induced phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. Trimucrin also reversed the DNA-binding activity of NF-κB by suppressing the LPS-induced nuclear translocation of p65 and the cytosolic IκB release. Flow cytometric analyses showed that trimucrin bound to cells in a concentration-dependent manner. The anti-αVβ3 mAb also specifically decreased the binding of fluorescein isothiocyanate (FITC)-conjugated trimucrin. Binding assays demonstrated that integrin αVβ3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin decreases the inflammatory reaction through the attenuation of iNOS expression and nitric oxide (NO) production by blocking MAP kinase and the NF-κB activation in LPS-stimulated THP-1 and RAW 264.7 cells.

  16. 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.

  17. An antimicrobial peptide with angiogenic properties, AG-30/5C, activates human mast cells through the MAPK and NF-κB pathways.

    PubMed

    Kanazawa, Kazo; Okumura, Ko; Ogawa, Hideoki; Niyonsaba, François

    2016-04-01

    Apart from their direct antimicrobial activities against invading pathogens, antimicrobial peptides exhibit additional protective functions that have led to their being named host defense peptides (HDPs). These functions include the stimulation of the production of cytokines/chemokines, the promotion of chemotaxis and cell proliferation and the induction of angiogenesis and wound healing. AG-30/5C is a novel angiogenic HDP that in addition to its antimicrobial activity also activates fibroblasts and endothelial cells and promotes angiogenesis and wound healing. Given that mast cells are found primarily in the vicinity of vessels, where they are intimately involved in wound healing, we hypothesized that AG-30/5C may activate mast cells. We demonstrated that AG-30/5C activated LAD2 human mast cells to degranulate and produce lipid mediators including leukotriene C4, prostaglandin D2 and E2. Moreover, AG-30/5C increased mast cell chemotaxis and induced the production of the cytokines GM-CSF and TNF-α and various chemokines, such as IL-8, MCP-1, MCP-3, MIP-1α and MIP-1β. The chemotaxis and cytokine/chemokine production induced by AG-30/5C were suppressed by both pertussis toxin and U-73122, suggesting the involvement of the G protein and phospholipase C pathways in AG-30/5C-induced mast cell activation. Furthermore, these pathways were activated downstream of the MAPK and NF-κB signaling molecules, as demonstrated by the inhibitory effects of ERK-, JNK-, p38- and NF-κB-specific inhibitors on cytokine/chemokine production. Interestingly, AG-30/5C caused the phosphorylation of MAPKs and IκB. We suggest that the angiogenic and antimicrobial peptide AG-30/5C plays a key role in the recruitment and activation of human mast cells at inflammation and wound sites.

  18. Lipopolysaccharide induced LOX-1 expression via TLR4/MyD88/ROS activated p38MAPK-NF-κB pathway.

    PubMed

    Zhao, Wenwen; Ma, Guixin; Chen, Xiuping

    2014-12-01

    Lectin-like receptor for oxidized low density lipoprotein (LOX-1) plays a key role in endothelial ox-LDL endocytosis, endothelial dysfunction and atherogenesis. In the present study, the effect of lipopolysaccharide (LPS) on LOX-1 expression and the underlying molecular pathways were investigated. Human umbilical vein endothelial cells (HUVECs) were treated with LPS and the protein expressions of LOX-1, TLR4, TLR2, MyD88, Nox4, Nox2, PI3K, p38MAPK, JNK, ERK, Nrf1, Nrf2 and p65 were examined by Western blotting. The intracellular reactive oxygen species (ROS) production was examined by flow cytometry with fluorescence probe DCFH2-DA. The role of TLR4, MyD88 and Nox4 were determined with specific siRNA. The endothelial ox-LDL uptake and the endothelial-monocyte adhesion were evaluated with DiI-ox-LDL and Hoechst 33342 respectively. The effect of LPS on LOX-1 expression in aorta tissue was also studied with male C57/BL6 mice by intraperitoneal injection of LPS. The results showed that LPS induced LOX-1 protein expression in a time- and concentration-dependent manner. The mRNA expression of LOX-1 was also upregulated. The protein expression of LOX-1 and phosphorylated p38MAPK, p65 was significantly enhanced by LPS both in vitro and in vivo. LPS induced LOX-1 expression was blocked by siRNA for TLR4, MyD88, and Nox4 and inhibitors for p38MAPK, NF-κB, cyclooxygenase-2, and NADPH oxidase. Both LPS induced ox-LDL uptake and endothelial-monocyte adhesion were significantly inhibited by anti-LOX-1 antibody. LPS dramatically induced LOX-1 protein expression in aorta tissues. In conclusion, our data suggested that LPS induces LOX-1 expression via TLR4/MyD88/ROS activated p38MAPK/NF-κB pathway in endothelial cells, which provides new regulatory mechanisms for LOX-1 expression.

  19. 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.

  20. Status of Japanese Experiment Module (JEM) activities

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The current status of the JEM activities are presented in graphic form. The JEM spacecraft configuration is presented. The JEM configuration consist of the Pressurized Module, the Exposed Facility, the Experiment Logistics Module which consist of a pressurized section and an exposed section; and the Remote Manipulator System. The master schedule of the space station is given. Also the development tests of the structure and mechanism, the electrical power system, the data management system, the thermal control system, the environment control system, the experiment support system, and the remote manipulator system are listed.

  1. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway.

    PubMed

    Tanaka, Yuichi; Gavrielides, M Veronica; Mitsuuchi, Yasuhiro; Fujii, Teruhiko; Kazanietz, Marcelo G

    2003-09-05

    Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. BRAF mutations and phosphorylation status of mitogen-activated protein kinases in the development of flat and depressed-type colorectal neoplasias.

    PubMed

    Konishi, K; Takimoto, M; Kaneko, K; Makino, R; Hirayama, Y; Nozawa, H; Kurahashi, T; Kumekawa, Y; Yamamoto, T; Ito, H; Yoshikawa, N; Kusano, M; Nakayama, K; Rembacken, B J; Ota, H; Imawari, M

    2006-01-30

    Although some molecular differences between flat-depressed neoplasias (FDNs) and protruding neoplasias (PNs) have been reported, it is uncertain if the BRAF mutations or the status of phosphorylated mitogen-activated protein kinase (p-MAPK) are different between theses two groups. We evaluated the incidence of BRAF and KRAS mutations, high-frequency microsatellite instability (MSI-H), and the immunohistochemical status of p-MAPK in the nonserrated neoplasias (46 FDNs and 57 PNs). BRAF mutations were detected in four FDNs (9%) and none of PNs (P=0.0369 by Fisher's exact test). KRAS mutations were observed in none of FDNs and in 14 PNs (25%; P=0.0002 by Fisher's exact test). MSI-H was detected in seven out of 44 FDNs (16%) and in one out of 52 of PNs (2%) (P=0.022 by Fisher's exact test). Type B and C immunostaining for p-MAPK was observed in 34 out of 46 FDNs (72%), compared with 24 out of 55 PNs (44%; P=0.0022 by chi(2) test). There was no significant difference in the type B and C immunostaining of p-MAPK between FDNs with and without BRAF mutations. BRAF and KRAS mutations are mutually exclusive in the morphological characteristics of colorectal nonserrated neoplasia. Abnormal accumulation of p-MAPK protein is more likely to be implicated in the tumorigenesis of FDNs than of PNs. However, this abnormality in FDNs might occur via the genetic alteration other than BRAF or KRAS mutation.

  4. Flavonoids Identified from Korean Scutellaria baicalensis Georgi Inhibit Inflammatory Signaling by Suppressing Activation of NF-κB and MAPK in RAW 264.7 Cells

    PubMed Central

    Hong, Gyeong-Eun; Kim, Jin-A.; Nagappan, Arulkumar; Yumnam, Silvia; Lee, Ho-Jeong; Kim, Eun-Hee; Lee, Won-Sup; Shin, Sung-Chul; Park, Hyeon-Soo; Kim, Gon-Sup

    2013-01-01

    Scutellaria baicalensis Georgi has been used as traditional medicine for treating inflammatory diseases, hepatitis, tumors, and diarrhea in Asia. Hence, we investigated the anti-inflammatory effect and determined the molecular mechanism of action of flavonoids isolated from Korean S. baicalensis G. in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine cytotoxicity of the flavonoids at various concentrations of 10, 40, 70, and 100 µg/mL. No cytotoxicity was observed in RAW 264.7 cells at these concentrations. Furthermore, the flavonoids decreased production of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-alpha and inhibited phosphorylation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells. Moreover, to identify the differentially expressed proteins in RAW 264.7 cells of the control, LPS-treated, and flavonoid-treated groups, two-dimensional gel electrophoresis and mass spectrometry were conducted. The identified proteins were involved in the inflammatory response and included PRKA anchor protein and heat shock protein 70 kD. These findings suggest that the flavonoids isolated from S. baicalensis G. might have anti-inflammatory effects that regulate the expression of inflammatory mediators by inhibiting the NF-κB signaling pathway via the MAPK signaling pathway in RAW 264.7 cells. PMID:24348728

  5. 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

  6. EGF receptor-dependent mechanism may be involved in the Tamm-Horsfall glycoprotein-enhanced PMN phagocytosis via activating Rho family and MAPK signaling pathway.

    PubMed

    Li, Ko-Jen; Siao, Sue-Cien; Wu, Cheng-Han; Shen, Chieh-Yu; Wu, Tsai-Hung; Tsai, Chang-Youh; Hsieh, Song-Chou; Yu, Chia-Li

    2014-01-21

    Our previous studies showed that urinary Tamm-Horsfall glycoprotein (THP) potently enhanced polymorphonuclear neutrophil (PMN) phagocytosis. However, the domain structure(s), signaling pathway and the intracellular events responsible for THP-enhanced PMN phagocytosis remain to be elucidated. THP was purified from normal human urine. The human promyelocytic leukemia cell line HL-60 was induced to differentiate into PMNs by all-trans retinoid acid. Pretreatment with different MAPK and PI3K inhibitors was used to delineate signaling pathways in THP-enhanced PMN phagocytosis. Phosphorylation of molecules responsible for PMN phagocytosis induced by bacterial lipopolysaccharide (LPS), THP, or human recombinant epidermal growth factor (EGF) was evaluated by western blot. A p38 MAPK inhibitor, SB203580, effectively inhibited both spontaneous and LPS- and THP-induced PMN phagocytosis. Both THP and LPS enhanced the expression of the Rho family proteins Cdc42 and Rac that may lead to F-actin re-arrangement. Further studies suggested that THP and EGF enhance PMN and differentiated HL-60 cell phagocytosis in a similar pattern. Furthermore, the EGF receptor inhibitor GW2974 significantly suppressed THP- and EGF-enhanced PMN phagocytosis and p38 and ERK1/2 phosphorylation in differentiated HL-60 cells. We conclude that EGF receptor-dependent signaling may be involved in THP-enhanced PMN phagocytosis by activating Rho family and MAP kinase.

  7. Osteoinductive LIM Mineralization Protein-1 Suppresses Activation of NF-κB and Selectively Regulates MAPK Pathways in Pre-osteoclasts

    PubMed Central

    Liu, Hui; Bargouti, Maggie; Zughaier, Susu; Zheng, Zhaomin; Liu, Yunshan; Sangadala, Sreedhara; Boden, Scott D.; Titus, Louisa

    2009-01-01

    LIM Mineralization Protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible Nitric Oxide Synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-κB), and prevented the phosphorylation of inhibitor of kappa B (IκB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-κB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-κB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways. PMID:19931434

  8. Osteoinductive LIM mineralization protein-1 suppresses activation of NF-kappaB and selectively regulates MAPK pathways in pre-osteoclasts.

    PubMed

    Liu, Hui; Bargouti, Maggie; Zughaier, Susu; Zheng, Zhaomin; Liu, Yunshan; Sangadala, Sreedhara; Boden, Scott D; Titus, Louisa

    2010-05-01

    LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible nitric oxide synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-kappaB), and prevented the phosphorylation of inhibitor of kappa B (IkappaB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-kappaB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-kappaB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways.

  9. Mucroporin-M1 Inhibits Hepatitis B Virus Replication by Activating the Mitogen-activated Protein Kinase (MAPK) Pathway and Down-regulating HNF4α in Vitro and in Vivo*

    PubMed Central

    Zhao, Zhenhuan; Hong, Wei; Zeng, Zhengyang; Wu, Yingliang; Hu, Kanghong; Tian, Xiaohui; Li, Wenxin; Cao, Zhijian

    2012-01-01

    Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources. PMID:22791717

  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. Megakaryocytic Maturation in Response to Shear Flow Is Mediated by the Activator Protein 1 (AP-1) Transcription Factor via Mitogen-activated Protein Kinase (MAPK) Mechanotransduction.

    PubMed

    Luff, Stephanie A; Papoutsakis, Eleftherios T

    2016-04-08

    Megakaryocytes (MKs) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation to release pro/preplatelets into circulating blood. Shear forces promote DNA synthesis, polyploidization, and maturation in MKs, and platelet biogenesis. To investigate mechanisms underlying these MK responses to shear, we carried out transcriptional analysis on immature and mature stem cell-derived MKs exposed to physiological shear. In immature (day (d)9) MKs, shear exposure up-regulated genes related to growth and MK maturation, whereas in mature (d12) MKs, it up-regulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, six activator protein 1 (AP-1) transcripts (ATF4,JUNB,JUN,FOSB,FOS, andJUND) were up-regulated at d9 and two AP-1 proteins (JunD and c-Fos) were up-regulated both at d9 and d12. We show that mitogen-activated protein kinase (MAPK) signaling is linked to both the shear stress response and AP-1 up-regulation. c-Jun N-terminal kinase (JNK) phosphorylation increased significantly following shear stimulation, whereas JNK inhibition reduced shear-induced JunD expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos expression. JNK inhibition reduced fibrinogen binding and P-selectin expression of d12 platelet-like particles (PLPs), whereas p38 inhibition reduced fibrinogen binding of d12 PLPs. AP-1 expression correlated with increased MK DNA synthesis and polyploidization, which might explain the observed impact of shear on MKs. To summarize, we show that MK exposure to shear forces results in JNK activation, AP-1 up-regulation, and downstream transcriptional changes that promote maturation of immature MKs and platelet biogenesis in mature MKs.

  12. Activation of p44/42 MAPK plays a role in the TBT-induced loss of human natural killer (NK) cell function.

    PubMed

    Dudimah, Fred D; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M

    2010-10-01

    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 Toxicology 209:263-277, 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 ((51)Cr 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 sixfold increase in phospho-p44/42 levels. Previous studies showed a fivefold 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.

  13. Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil.

    PubMed

    Gao, Jing; Gao, Jin; Qian, Lan; Wang, Xia; Wu, Mingyuan; Zhang, Yang; Ye, Hao; Zhu, Shunying; Yu, Yan; Han, Wei

    2014-08-01

    Chemotherapy-induced mucositis (CIM) is a major does limiting side-effect of chemoagents such as 5-fluorouracil (5-FU). Molecules involved in this disease process are still not fully understood. We proposed that the homeostatically regulated genes during CIM may participate in the disease. A cluster of such genes were previously identified by expression gene-array from the mouse jejunum in 5-FU-induced mucositis model. Here, we report that CXCL4 is such a homeostatically regulated gene and serves as a new target for the antibody treatment of CIM. CXCL4 and its receptor CXCR3 were confirmed at both the gene and protein levels to be homeostatically regulated during 5-FU-induced mucositis. Using of CXCL4 neutralizing monoclonal antibody (CXCL4mab) decreased the incidence, severity, and duration of the chemotherapy-induced diarrhea, the major symptom of CIM, in a 5-FU mouse CIM model. Mechanistically, CXCL4mab reduced the apoptosis of the crypt epithelia by suppression of the 5-FU-induced expression of p53 and Bax through its receptor CXCR3. The downstream signaling pathway of CXCL4 in activation of the epithelial apoptosis was identified in an intestinal epithelial cell line (IEC-6). CXCL4 activated the phosphorylation of p38 MAPK, which mediated the stimulated expression of p53 and Bax, and resulted in the ultimate activation of Caspase-8, -9, and -3. Taken together, activation of CXCL4 expression by 5-FU in mice participates in 5-FU-induced intestinal mucositis through upregulation of p53 via activation of p38-MAPK, and CXCL4mab is potentially beneficial in preventing CIM in the intestinal tract.

  14. Resveratrol Suppresses Cytokine Production Linked to FcεRI-MAPK Activation in IgE-Antigen Complex-Exposed Basophilic Mast Cells and Mice.

    PubMed

    Han, Seon-Young; Choi, Yean-Jung; Kang, Min-Kyung; Park, Jung Han Yoon; Kang, Young-Hee

    2015-01-01

    A complicated interplay between resident mast cells and other recruited inflammatory cells contributes to the development and progression of allergic inflammation entailing the promotion of T helper 2 (Th2) cytokine responses. The current study examined whether resveratrol suppressed the production of inflammatory Th2 cytokines in cultured rat basophilic leukemia RBL-2H3 cells. Cells pre-treated with resveratrol nontoxic at 1–25 μM were sensitized with anti-dinitrophenyl (anti-DNP), and subsequently stimulated by dinitrophenyl-human serum albumin (DNP–HSA) antigen. Resveratrol dose-dependently diminished the secretion of interleukin (IL)-3, IL-4, IL-13 as well as tumor necrosis factor (TNF)-α by the antigen stimulation from sensitized cells. It was found that resveratrol mitigated the phosphorylation of p38 MAPK, ERK, and JNK elevated in mast cells exposed to Fc epsilon receptor I (FcεRI)-mediated immunoglobulin E (IgE)-antigen complex. The FcεRI aggregation was highly enhanced on the surface of mast cells following the HSA stimulation, which was retarded by treatment with 1–25 μM resveratrol. The IgE-receptor engagement rapidly induced tyrosine phosphorylation of c-Src-related focal adhesion protein paxillin involved in the cytoskeleton rearrangement. The FcεRI-mediated rapid activation of c-Src and paxillin was attenuated in a dose-dependent manner. In addition, the paxillin activation entailed p38 MAPK and ERK-responsive signaling, but the JNK activation was less involved. Consistently, oral administration of resveratrol reduced the tissue level of phosphorylated paxillin in the dorsal skin of DNP–HSA-challenged mice. The other tyrosine kinase Tyk2-STAT1 signaling was activated in the dorsal epidermis of antigen-exposed mice, which was associated with allergic inflammation. These results showed that resveratrol inhibited Th2 cytokines- and paxillin-linked allergic responses dependent upon MAPK signaling. Therefore, resveratrol may possess the

  15. Telmisartan ameliorates cisplatin-induced nephrotoxicity by inhibiting MAPK mediated inflammation and apoptosis.

    PubMed

    Malik, Salma; Suchal, Kapil; Gamad, Nanda; Dinda, Amit Kumar; Arya, Dharamvir Singh; Bhatia, Jagriti

    2015-02-05

    Nephrotoxicity is a major adverse effect of the widely used anticancer drug cisplatin. Oxidative stress, inflammation and apoptosis are implicated in the pathophysiology of cisplatin-induced acute renal injury. Moreover, cisplatin activates many signal transduction pathways involved in cell injury and death, particularly mitogen activated protein kinase (MAPK) pathway. With this background, we aimed to investigate the protective effect of telmisartan, a widely used antihypertensive drug, in cisplatin-induced nephrotoxicity model in rats. To accomplish this, male albino wistar rats (150-200 g) were divided into 6 groups: Normal, cisplatin-control, telmisartan (2.5, 5 and 10 mg/kg) and telmisartan per se treatment groups. Normal saline or telmisartan was administered orally to rats for 10 days and cisplatin was given on 7th day (8 mg/kg; i.p.) to induce nephrotoxicity. On 10th day, rats were killed and both the kidneys were harvested for biochemical, histopathological and molecular studies. Cisplatin injected rats showed depressed renal function, altered proxidant-antioxidant balance and acute tubular necrosis which was significantly normalized by telmisartan co-treatment. Furthermore, cisplatin administration activated MAPK pathway that caused tubular inflammation and apoptosis in rats. Telmisartan treatment significantly prevented MAPK mediated inflammation and apoptosis. Among the three doses studied telmisartan at 10 mg/kg dose showed maximum nephroprotective effect which could be due to maintenance of cellular redox status and inhibition of MAPK activation.

  16. Phellinus linteus Extract Exerts Anti-asthmatic Effects by Suppressing NF-κB and p38 MAPK Activity in an OVA-induced Mouse Model of Asthma

    PubMed Central

    Yan, Guang Hai

    2014-01-01

    Phellinus linteus has been used as a traditional herbal medicine in Asian countries and is known to have anti-tumor, immunomodulatory, anti-inflammatory, and anti-allergic activities. However, the protective effects of P. linteus against experimental asthma have not been fully investigated. The objective of this study was to determine whether P. linteus ethanol extract (PLE) suppresses inflammatory response in an OVA-induced asthma model. As expected, the oral administration of PLE significantly inhibited eosinophilic airway inflammation and airway hyperresponsiveness in OVA-challenged BALB/c mice. Supporting these data, the augmentation of Th2 cytokines (IL-4, IL-5, and IL-13), eotaxin, and adhesion molecules in lung tissues and bronchoalveolar lavage fluid after OVA inhalation was markedly attenuated by PLE. Furthermore, PLE reduced OVA-induced activation of NF-κB and p38 MAPK in lung tissues. Therefore, our results suggest the potential of P. linteus as a therapeutic agent for asthma. PMID:24851100

  17. 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

  18. Genkwanin Inhibits Proinflammatory Mediators Mainly through the Regulation of miR-101/MKP-1/MAPK Pathway in LPS-Activated Macrophages

    PubMed Central

    Gao, Yuan; Liu, Fen; Fang, Lei; Cai, Runlan; Zong, Chuanjie; Qi, Yun

    2014-01-01

    Genkwanin is one of the major non-glycosylated flavonoids in many herbs with anti-inflammatory activities. Although its anti-inflammatory activity in vivo has been reported, the potential molecular mechanisms remain obscure. In this study, by pharmacological and genetic approaches, we explore the anti-inflammatory effects of genkwanin in LPS-activated RAW264.7 macrophages. Genkwanin potently decreases the proinflammatory mediators, such as iNOS, TNF-α, IL-1β and IL-6, at the transcriptional and translational levels without cytotoxicity, indicating the excellent anti-inflammatory potency of genkwanin in vitro. Mechanism study shows that genkwanin significantly suppresses the p38- and JNK-mediated AP-1 signaling pathway and increases the mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression at the posttranscriptional level. We also confirmed that microRNA-101 (miR-101) is a negative regulator of MKP-1 expression. Moreover, regardless of miR-101-deficient cells or miR-101-abundant cells, the suppression effects of genkwanin on supernatant proinflammatory mediators' levels are far less than that in respective negative control cells, suggesting that genkwanin exerts anti-inflammatory effect mainly through reducing miR-101 production. However, genkwanin can't affect the level of phospho-Akt (p-Akt), indicating that the phosphorylation of Akt may be not responsible for the effect of genkwanin on miR-101 production. We conclude that genkwanin exerts its anti-inflammatory effect mainly through the regulation of the miR-101/MKP-1/MAPK pathway. PMID:24800851

  19. 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

  20. 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

  1. 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

  2. Correlation of prolactin levels and PRL-receptor expression with Stat and Mapk cell signaling in the prostate of long-term sexually active rats.

    PubMed

    Rojas-Durán, Fausto; Pascual-Mathey, Luz I; Serrano, Karina; Aranda-Abreu, Gonzalo E; Manzo, Jorge; Soto-Cid, Abraham H; Hernandez, Ma Elena

    2015-01-01

    Prolactin (PRL) is a key hormone for prostate function, with a basal level in serum and associated with two characteristic circadian peaks. In the male rat, the execution of one bout of sexual behavior with consecutive ejaculations produces a significant transient increase in PRL. However, the impact of a constant sexual life on both PRL levels and prostate function is unknown. Thus, by using constantly copulating males we analyzed the levels of serum PRL, the effect on prostate PRL receptors, and activation of pStat3, pStat5 and Mapk signaling pathways. Sexually experienced Wistar male rats were used, which underwent periodic sessions of sexual behavior tests. Males were subjected to a session of sexual behavior to achieve at least one and up to four ejaculations. Of these, a blood sample was collected from randomly selected males and the ventral prostate was removed for analysis. Serum PRL was quantified, the mRNA for PRL receptors was determined, and signaling pathways were analyzed. Data show that a constant sexual life produced a constant elevation of PRL in serum during four consecutive ejaculations. The ventral prostate showed a different mRNA expression profile for the long and short isoform of the PRL receptor, and both mRNA levels increased. Although the gland did not show modification of the activation of the pStat5 signaling pathway, the levels of pStat3 increased, and the Mapk pathway showed one significant elevation after the third ejaculation. Thus, we showed that an active and constant sexual life produces a sustained increase in serum PRL, its receptors, and the pStat3 signaling pathway. These responses seem to underlie the required physiological need to produce the quantity and quality of prostatic semen to ensure the appropriate environment for sperm to reach and fertilize the ovum.

  3. P2Y12 receptor-mediated activation of spinal microglia and p38MAPK pathway contribute to cancer-induced bone pain

    PubMed Central

    Liu, Mingjuan; Yao, Ming; Wang, Hanqi; Xu, Longsheng; Zheng, Ying; Huang, Bing; Ni, Huadong; Xu, Shijie; Zhou, Xuyan; Lian, Qingquan

    2017-01-01

    Background Cancer-induced bone pain (CIBP) is one of the most challenging clinical problems due to a lack of understanding the mechanisms. Recent evidence has demonstrated that activation of microglial G-protein-coupled P2Y12 receptor (P2Y12R) and proinflammatory cytokine production play an important role in neuropathic pain generation and maintenance. However, whether P2Y12R is involved in CIBP remains unknown. Methods The purpose of this study was to investigate the role of P2Y12R in CIBP and its molecular mechanisms. Using the bone cancer model inoculated with Walker 256 tumor cells into the left tibia of Sprague Dawley rat, we blocked spinal P2Y12R through intrathecal administration of its selective antagonist MRS2395 (400 pmol/µL, 15 µL). Results We found that not only the ionized calcium-binding adapter molecule 1 (Iba-1)-positive microglia in the ipsilateral spinal cord but also mechanical allodynia was significantly inhibited. Furthermore, it decreased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and the production of proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6), whereas it increased tumor necrosis factor-α (TNF-α). Conclusion Taken together, our present results suggest that microglial P2Y12R in the spinal cord may contribute to CIBP by the activation of spinal microglia and p38MAPK pathway, thus identifying a potential therapeutic target for the treatment of CIBP. PMID:28243146

  4. Role of VDR in 1α,25-dihydroxyvitamin D3-dependent non-genomic activation of MAPKs, Src and Akt in skeletal muscle cells.

    PubMed

    Buitrago, Claudia; Pardo, Verónica Gonzalez; Boland, Ricardo

    2013-07-01

    1α,25-dihydroxyvitamin D3 [1,25D] is recognized as a steroid hormone that rapidly elicits intracellular signals in various tissues. In skeletal myoblasts, we have previously demonstrated that one of the 1,25D-induced non-genomic effects is the upstream stimulation of MAPKs through Src activation. In this work, the data obtained suggest that the classical receptor of vitamin D (VDR) participates in non-transcriptional actions of 1,25D. We significantly reduced VDR expression by infection of C2C12 murine myoblasts with lentiviral particles containing the pLKO.1 plasmid with information to express a shRNA against mouse VDR. In these cells (C2C12-shVDR), Western blot analyses show that 1,25D-induced p38 MAPK activation and Src tyr416 phosphorylation were abolished. In addition, 1,25D-dependent activity of ERK1/2 was diminished in cells lacking VDR but to a lesser extent (∼-60%). Phosphorylation of Akt by 1,25D, recently demonstrated in C2C12 cells, in the present work also appeared to be partially dependent on VDR expression (∼50% in C2C12-shVDR cells). Our results indicate that VDR is involved in 1,25D-induced rapid events related to survival/proliferation responses in skeletal muscle cells, providing relevant information on the mechanism of initiation of the non-genomic hormone signal. The participation of a VDR-independent non-genomic mechanism of action should also be taken into consideration. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

  5. 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.

  6. Ligation of CD47 induces G1 arrest in EBV-transformed B cells through ROS generation, p38 MAPK/JNK activation, and Tap73 upregulation.

    PubMed

    Park, Ga Bin; Bang, Si Ra; Lee, Hyun-Kyung; Kim, Daejin; Kim, Seonghan; Kim, Jin Kyoung; Kim, Yeong Seok; Hur, Dae Young

    2014-01-01

    CD47 is expressed in normal activated cells as well as in several tumors. It also has been implicated as having antiangiogenic and antimetastatic properties, but its roles in Epstein-Barr virus (EBV)-transformed B cells are still not fully understood. Herein, we report that EBV infection induced CD47 surface expression on B cells, and CD47 ligation with anti-CD47 mAb (B6H12) reduced cell proliferation and induced G1 arrest. CD47-induced G1 arrest was mediated through increased cyclin-dependent kinase inhibitors (CDKi) and a simultaneously decreased CDK/cyclins, and p38 MAPK/JNK activation preceded binding of CDKi-CDK. Moreover, reactive oxygen species (ROS) generation and upregulation of both TAp73 and ER stress sensor proteins were detected after CD47 ligation, and p38 inhibitor SB203580 and JNK inhibitor SP600125 blocked upregulation of TAp73 and cell cycle arrest. We investigated whether ROS generation is the initial event of CD47-mediated G1 arrest because ROS scavenger NAC effectively abrogated the majority of CD47-mediated responses but SB203580 and SP600125 did not block ROS production. Taken together, we concluded that CD47 ligation on EBV-transformed B cells led to G1 arrest by ROS generation and, subsequently, there was p38 MAPK/JNK pathway activation, ER stress triggering, and TAp73 upregulation. Our findings provide data supporting CD47 as a feasible target for EBV-associated tumor therapy.

  7. 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

  8. Mechanism of action of the breast cancer-promoter hormone, 5α-dihydroprogesterone (5αP), involves plasma membrane-associated receptors and MAPK activation.

    PubMed

    Wiebe, John P; Pawlak, Kevin J; Kwok, Arthur

    2016-01-01

    Previous studies have shown that breast tissues and breast cell lines can convert progesterone to 5α-pregnane-3,20-dione (5aP), and that 5αP stimulates breast cell proliferation and detachment in vitro, and tumor formation in vivo, regardless of presence or absence of receptors for progesterone (PR) or estrogen (ER). Recently it was demonstrated, both in vitro and in vivo, that pro-cancer actions attributed to administered progesterone are due to the in situ produced 5αP. Because of the significant role of 5αP in breast cancers, it is important to understand its molecular mechanisms of action. The aims of the current studies were to identify 5αP binding sites and to determine if the mechanisms of action of 5αP involve the mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinases (ERK1/2) pathway. Binding studies, using tritium-labeled 5αP ([(3)H]5αP), carried out on membrane, cytosol and nuclear fractions from human breast cells (MCF-7, PR/ER-positive; MDA-MB-231, PR/ER-negative) and on highly enriched membrane fractions, identified the plasma membrane as the site of ligand specific 5αP receptors. Localization of 5αP receptors to the cell membrane was confirmed visually with fluorescently labeled conjugate (5αP-BSA-FITC). Treatment of cells with either 5αP or membrane-impermeable 5αP-BSA resulted in significant increases in cell proliferation and detachment. 5αP and 5αP-BSA equally activated the MAPK/ERK1/2 pathway as evidenced by phosphorylation of ERK1/2. Inhibitors (PD98059, mevastatin and genistein) of specific sites along the Ras/Raf/MEK/ERK signaling pathway, blocked the phosphorylation and concomitantly inhibited 5αP-induced stimulation of cell proliferation and detachment. The study has identified high affinity, stereo-specific binding sites for 5αP that have the characteristics of a functional membrane 5αP receptor, and has shown that the cancer-promoter actions of 5αP are mediated from the liganded receptor

  9. Tartary buckwheat flavonoids protect hepatic cells against high glucose-induced oxidative stress and insulin resistance via MAPK signaling pathways.

    PubMed

    Hu, Yuanyuan; Hou, Zuoxu; Liu, Dongyang; Yang, Xingbin

    2016-03-01

    Oxidative stress plays a crucial role in chronic complication of diabetes. In this study, the protective effect of purified tartary buckwheat flavonoids (TBF) fraction against oxidative stress induced by a high-glucose challenge, which causes insulin resistance, was investigated on hepatic HepG2 cells. Oxidative status, phosphorylated mitogen-activated protein kinases (MAPKs), nuclear factor E2 related factor 2 (Nrf2) and p-(Ser307)-IRS-1 expression, and glucose uptake were evaluated. Results suggest that treatment of HepG2 cells with TBF alone improved glucose uptake and antioxidant enzymes, and activated Nrf2, and attenuated the IRS-1 Ser307 phosphorylation, and enhanced total levels of IRS-1. Furthermore, the high glucose-induced changes in antioxidant defences, Nrf2, p-MAPKs, p-IRS1 Ser307, and IRS-1 levels, and glucose uptake were also significantly inhibited by pre-treatment with TBF. Interestingly, the selective MAPK inhibitors significantly enhanced the TBF-mediated protection by inducing changes in the redox status, glucose uptake, p-(Ser307) and total IRS-1 levels. This report firstly showed that TBF could recover the redox status of insulin-resistant HepG2 cells, suggesting that TBF significantly protected the cells against high glucose-induced oxidative stress, and these beneficial effects of TBF on redox balance and insulin resistance were mediated by targeting MAPKs.

  10. Anti-Inflammatory Activities of Inotilone from Phellinus linteus through the Inhibition of MMP-9, NF-κB, and MAPK Activation In Vitro and In Vivo

    PubMed Central

    Huang, Guan-Jhong; Huang, Shyh-Shyun; Deng, Jeng-Shyan

    2012-01-01

    Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK) [extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4th and the 5th h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5th h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5th h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT

  11. Differential immunotoxic effects of ethanol on murine EL-4 lymphoma and normal lymphocytes is mediated through increased ROS production and activation of p38MAPK.

    PubMed

    Premachandran, Sudha; Khan, Nazir M; Thakur, Vikas S; Shukla, Jyoti; Poduval, T B

    2012-08-01

    Ethanol has been used to achieve thymic depletion in myasthenia gravis patients. Ethanol (95%) has also been used widely in the therapy of many tumors including hepatocellular carcinoma. In light of these findings, we delineated the differential immunotoxic behavior and mechanism of lower concentration of ethanol towards murine EL-4 lymphoma and its normal counterpart lymphocytes. EL-4 lymphoma and normal lymphocytes were cultured with ethanol (0%-5%) for 6 h and cytotoxicity was measured by various methods. EL-4 cells treated with ethanol showed concentration-dependent loss of viability at 2%-5% ethanol concentration and exhibit proliferative arrest at preG1 stage. Acridine-orange and ethidium-bromide staining indicated that ethanol induced death in EL-4 cells, by induction of both apoptosis and necrosis which was further supported by findings of DNA-fragmentation and trypan blue dye exclusion test. However, treatment of lymphocytes with similar concentration of ethanol did not show any death-associated parameters. Furthermore, ethanol induced significantly higher ROS generation in EL-4 cells as compared to lymphocytes and caused PARP cleavage and activation of apoptotic proteins like p53 and Bax, in EL-4 cells and not in normal lymphocytes. In addition, ethanol exposure to EL-4 cells led to phosphorylation of p38MAPK, and upregulation of death receptor Fas (CD95). Taken together, these results suggest that ethanol upto a concentration of 5% caused no significant immunotoxicity towards normal lymphocytes and induced cell death in EL-4 cells via phosphorylation of p38MAPK and regulation of p53 leading to further activation of both extrinsic (Fas) and intrinsic (Bax) apoptotic markers.

  12. Latex of Euphorbia antiquorum-induced S-phase arrest via active ATM kinase and MAPK pathways in human cervical cancer HeLa cells.

    PubMed

    Hsieh, Wen-Tsong; Lin, Hui-Yi; Chen, Jou-Hsuan; Lin, Wen-Chung; Kuo, Yueh-Hsiung; Wood, W Gibson; Lu, Hsu-Feng; Chung, Jing-Gung

    2015-09-01

    Latex of Euphorbia antiquorum (EA) has demonstrated great chemotherapeutic potential for cancer. However, the mechanisms of anti-proliferation of EA on cancer cell remain to be further investigated. The purpose of this study was to explore the influence of EA in human cervical cancer cells. Here, the cell cycle distribution by flow cytometry was examined and the protein expression by the western blotting methods was analyzed. From the cytometric results it was shown that EA-induced S-phase arrest in a concentration manner both in human cervical cancer HeLa and CaSki cells. According the western blot results it was illustrated that EA could downregulate early cyclin E1-Cdk2; and cyclin A-Cdc2 provides a significant additional quantity of S-phase promotion, that in turn promoted the expression of p21(waf1/cip1) and p27(kip1) which were the inhibitors in the complex of cyclin A and Cdc2 that led to cell cycle arrest. Moreover, EA promoted the activation of ataxia telangiectasia mutated (ATM) and check-point kinase-2 (Chk2); however, it negatively regulated the expression of Topoisomerases I and II, Cdc25A, and Cdc25C signaling. Caffeine, an ATM/ATR inhibitor significantly reversed EA downregulation in the levels of Cdc25A. Furthermore, JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 both could reverse the EA upregulation of the protein of Chk2 level, significantly. This study, therefore, revealed that EA could downregulate topoisomerase, and activate ATM kinase, which then induce parallel Chk 1/2 and MAPK signaling pathways to promote the degradation of Cdc25A to induced S-phase arrest in human cervical cancer HeLa cells.

  13. Oncogenic BRAF fusions in mucosal melanomas activate the MAPK pathway and are sensitive to MEK/PI3K inhibition or MEK/CDK4/6 inhibition.

    PubMed

    Kim, H S; Jung, M; Kang, H N; Kim, H; Park, C-W; Kim, S-M; Shin, S J; Kim, S H; Kim, S G; Kim, E K; Yun, M R; Zheng, Z; Chung, K Y; Greenbowe, J; Ali, S M; Kim, T-M; Cho, B C

    2017-01-16

    Despite remarkable progress in cutaneous melanoma genomic profiling, the mutational landscape of primary mucosal melanomas (PMM) remains unclear. Forty-six PMMs underwent targeted exome sequencing of 111 cancer-associated genes. Seventy-six somatic nonsynonymous mutations in 42 genes were observed, and recurrent mutations were noted on eight genes, including TP53 (13%), NRAS (13%), SNX31 (9%), NF1 (9%), KIT (7%) and APC (7%). Mitogen-activated protein kinase (MAPK; 37%), cell cycle (20%) and phosphatidylinositol 3-kinase (PI3K)-mTOR (15%) pathways were frequently mutated. We biologically characterized a novel ZNF767-BRAF fusion found in a vemurafenib-refractory respiratory tract PMM, from which cell line harboring ZNF767-BRAF fusion were established for further molecular analyses. In an independent data set, NFIC-BRAF fusion was identified in an oral PMM case and TMEM178B-BRAF fusion and DGKI-BRAF fusion were identified in two malignant melanomas with a low mutational burden (number of mutation per megabase, 0.8 and 4, respectively). Subsequent analyses revealed that the ZNF767-BRAF fusion protein promotes RAF dimerization and activation of the MAPK pathway. We next tested the in vitro and in vivo efficacy of vemurafenib, trametinib, BKM120 or LEE011 alone and in combination. Trametinib effectively inhibited tumor cell growth in vitro, but the combination of trametinib and BKM120 or LEE011 yielded more than additive anti-tumor effects both in vitro and in vivo in a melanoma cells harboring the BRAF fusion. In conclusion, BRAF fusions define a new molecular subset of PMM that can be targeted therapeutically by the combination of a MEK inhibitor with PI3K or cyclin-dependent kinase 4/6 inhibitors.Oncogene advance online publication,16 January 2017; doi:10.1038/onc.2016.486.

  14. 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

  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. LZAP Inhibits p38 MAPK (p38) Phosphorylation and Activity by Facilitating p38 Association with the Wild-Type p53 Induced Phosphatase 1 (WIP1)

    PubMed Central

    An, Hanbing; Lu, Xinyuan; Liu, Dan; Yarbrough, Wendell G.

    2011-01-01

    LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation. PMID:21283629

  18. LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1).

    PubMed

    An, Hanbing; Lu, Xinyuan; Liu, Dan; Yarbrough, Wendell G

    2011-01-24

    LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.

  19. 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.

  20. Triterpenoid Saponin W3 from Anemone flaccida Suppresses Osteoclast Differentiation through Inhibiting Activation of MAPKs and NF-κB Pathways

    PubMed Central

    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

  1. PDK2 promotes chondrogenic differentiation of mesenchymal stem cells by upregulation of Sox6 and activation of JNK/MAPK/ERK pathway

    PubMed Central

    Wang, H.; Shan, X.B.; Qiao, Y.J.

    2017-01-01

    This study was undertaken to clarify the role and mechanism of pyruvate dehydrogenase kinase isoform 2 (PDK2) in chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were isolated from femurs and tibias of Sprague-Dawley rats, weighing 300-400 g (5 females and 5 males). Overexpression and knockdown of PDK2 were transfected into MSCs and then cell viability, adhesion and migration were assessed. Additionally, the roles of aberrant PDK2 in chondrogenesis markers SRY-related high mobility group-box 6 (Sox6), type ΙΙ procollagen gene (COL2A1), cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), type ΙX procollagen gene (COL9A2) and collagen type 1 alpha 1 (COL1A1) were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expressions of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and extracellular regulated protein kinase (ERK) were measured. Overexpressing PDK2 promoted cell viability, adhesion and inhibited cell migration in MSCs (all P<0.05). qRT-PCR assay showed a potent increase in the mRNA expressions of all chondrogenesis markers in response to overexpressing PDK2 (P<0.01 or P<0.05). PDK2 overexpression also induced a significant accumulation in mRNA and protein expressions of JNK, p38MAPK and ERK in MSCs compared to the control (P<0.01 or P<0.05). Meanwhile, silencing PDK2 exerted the opposite effects on MSCs. This study shows a preliminary positive role and potential mechanisms of PDK2 in chondrogenic differentiation of MSCs. It lays the theoretical groundwork for uncovering the functions of PDK2 and provides a promising basis for repairing cartilage lesions in osteoarthritis. PMID:28225870

  2. Chemotherapy-related cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs

    PubMed Central

    Barreto, Rafael; Waning, David L.; Gao, Hongyu; Liu, Yunlong; Zimmers, Teresa A.; Bonetto, Andrea

    2016-01-01

    Cachexia affects the majority of cancer patients, with currently no effective treatments. Cachexia is defined by increased fatigue and loss of muscle function resulting from muscle and fat depletion. Previous studies suggest that chemotherapy may contribute to cachexia, although the causes responsible for this association are not clear. The purpose of this study was to investigate the mechanism(s) associated with chemotherapy-related effects on body composition and muscle function. Normal mice were administered chemotherapy regimens used for the treatment of colorectal cancer, such as Folfox (5-FU, leucovorin, oxaliplatin) or Folfiri (5-FU, leucovorin, irinotecan) for 5 weeks. The animals that received chemotherapy exhibited concurrent loss of muscle mass and muscle weakness. Consistently with previous findings, muscle wasting was associated with up-regulation of ERK1/2 and p38 MAPKs. No changes in ubiquitin-dependent proteolysis or in the expression of TGFβ-family members were detected. Further, marked decreases in mitochondrial content, associated with abnormalities at the sarcomeric level and with increase in the number of glycolytic fibers were observed in the muscle of mice receiving chemotherapy. Finally, ACVR2B/Fc or PD98059 prevented Folfiri-associated ERK1/2 activation and myofiber atrophy in C2C12 cultures. Our findings demonstrate that chemotherapy promotes MAPK-dependent muscle atrophy as well as mitochondrial depletion and alterations of the sarcomeric units. Therefore, these findings suggest that chemotherapy potentially plays a causative role in the occurrence of muscle loss and weakness. Moreover, the present observations provide a strong rationale for testing ACVR2B/Fc or MEK1 inhibitors in combination with anticancer drugs as novel strategies aimed at preventing chemotherapy-associated muscle atrophy. PMID:27259276

  3. Maternal liver damage delays meiotic resumption in bovine oocytes through impairment of signalling cascades originated from low p38MAPK activity in cumulus cells.

    PubMed

    Tanaka, H; Takeo, S; Monji, Y; Kuwayama, T; Iwata, H

    2014-02-01

    The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

  4. A Novel Lipopeptide from Skin Commensal Activates TLR2/CD36-p38 MAPK Signaling to Increase Antibacterial Defense against Bacterial Infection

    PubMed Central

    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 lysine11 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. PMID:23472173

  5. 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.

  6. 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.

  7. 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.

  8. RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways.

    PubMed

    Fang, Evandro Fei; Zhang, Chris Zhi Yi; Fong, Wing Ping; Ng, Tzi Bun

    2012-04-01

    Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker's yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.

  9. ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway

    PubMed Central

    1996-01-01

    AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways. PMID:8707830

  10. 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

  11. 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.

  12. 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

  13. 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

    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.

  14. Anti-tumor activity of SL4 against breast cancer cells: induction of G2/M arrest through modulation of the MAPK-dependent p21 signaling pathway

    PubMed Central

    Wang, Li-Hui; Jiang, Xiao-Rui; Chen, Guo-Liang; Guo, Wei; Zhang, Jing-Yuan; Cui, Li-Juan; Li, Hua-Huan; Li, Meng; Liu, Xing; Yang, Jing-Yu; Wu, Chun-Fu

    2016-01-01

    SL4, a chalcone-based compound, has been shown to retard tumor invasion and angiogenesis by suppressing HIF1 activity and to induce apoptosis by promoting ROS release. Here, we report that SL4 is able to inhibit the proliferation of different types of breast cancer cell in vitro and in vivo by inducing G2/M cell cycle arrest. Our results showed that SL4 exhibited strong anti-proliferative activity in several human breast cancer cell lines, with IC50 values lower than 1.3 μM. Further studies indicated that SL4 induced G2/M arrest in these cell lines. Mechanistically, SL4 reduces the expression of cyclin A2 and cdc25C and decreases the activity of the cdc2/cyclin B1 complex. Notably, SL4 treatment resulted in an obvious increase in p21 mRNA and protein levels through activation of MAPK signaling pathways, but not the TGF-β pathway. SP600125 and PD98059, specific inhibitors of JNK kinase and ERK kinase, significantly blocked the SL4-induced G2/M phase arrest and upregulation of p21. Furthermore, SL4 suppressed the growth of established breast tumors in nude mice through upregulation of p21 and downregulation of cdc25C, and displayed a good safety profile. Taken together, these findings demonstrate the potential value of SL4 as a novel multi-target anti-tumor drug candidate. PMID:27819344

  15. MAPK genes interact with diet and lifestyle factors to alter risk of breast cancer: The Breast Cancer Health Disparities Study

    PubMed Central

    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 three 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 ER/PR strata. MAP3K9 was associated with breast cancer overall (PARTP=0.02) with strongest association among women with the highest IA ancestry (PARTP=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. PMID:25629224

  16. 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.

  17. Polydatin ameliorates Staphylococcus aureus-induced mastitis in mice via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB pathway

    PubMed Central

    Jiang, Kang-feng; Zhao, Gan; Deng, Gan-zhen; Wu, Hai-chong; Yin, Nan-nan; Chen, Xiu-ying; Qiu, Chang-wei; Peng, Xiu-li

    2017-01-01

    Recent studies show that Polydatin (PD) extracted from the roots of Polygonum cuspidatum Sieb, a widely used traditional Chinese remedies, possesses anti-inflammatory activity in several experimental models. In this study, we investigated the anti-inflammatory effects of PD on Staphylococcus aureus-induced mastitis in mice and elucidated the potential mechanisms. In mice with S aureus-induced mastitis, administration of PD (15, 30, 45 mg/kg, ip) or dexamethasone (Dex, 5 mg/kg, ip) significantly suppressed the infiltration of inflammatory cells, ameliorated the mammary structural damage, and inhibited the activity of myeloperoxidase, a biomarker of neutrophils accumulation. Furthermore, PD treatment dose-dependently decreased the levels of TNF-α, IL-1β, IL-6 and IL-8 in the mammary gland tissues. PD treatment also dose-dependently decreased the expression of TLR2, MyD88, IRAK1, IRAK4 and TRAF6 as well as the phosphorylation of TAK1, MKK3/6, p38 MAPK, IκB-α and NF-κB in the mammary gland tissues. In mouse mammary epithelial cells (mMECs) infected by S aureus in vitro, pretreatment with PD dose-dependently suppressed the upregulated pro-inflammatory cytokines and signaling proteins, and the nuclear translocation of NF-κB p65 and AP-1. A TLR2-neutralizing antibody mimicked PD in its suppression on S aureus-induced upregulation of MyD88, p-p38 and p-p65 levels in mMECs. PD (50, 100 μg/mL) affected neither the growth of S aureus in vitro, nor the viability of mMECs. In conclusion, PD does not exhibit antibacterial activity against S aureus, its therapeutic effects in mouse S aureus-induced mastitis depend on its ability to down-regulate pro-inflammatory cytokine levels via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB signaling pathway. PMID:27890916

  18. 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

  19. Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38

    PubMed Central

    Grossi, Valentina; Lucarelli, Giuseppe; Forte, Giovanna; Peserico, Alessia; Matrone, Antonio; Germani, Aldo; Rutigliano, Monica; Stella, Alessandro; Bagnulo, Rosanna; Loconte, Daria; Galleggiante, Vanessa; Sanguedolce, Francesca; Cagiano, Simona; Bufo, Pantaleo; Trabucco, Senia; Maiorano, Eugenio; Ditonno, Pasquale; Battaglia, Michele; Resta, Nicoletta; Simone, Cristiano

    2015-01-01

    Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To gain insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in an STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients. PMID:26391455

  20. 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

  1. Alleviation of Microglial Activation Induced by p38 MAPK/MK2/PGE2 Axis by Capsaicin: Potential Involvement of other than TRPV1 Mechanism/s.

    PubMed

    Bhatia, Harsharan S; Roelofs, Nora; Muñoz, Eduardo; Fiebich, Bernd L

    2017-12-01

    Exaggerated inflammatory responses in microglia represent one of the major risk factors for various central nervous system's (CNS) associated pathologies. Release of excessive inflammatory mediators such as prostaglandins and cytokines are the hallmark of hyper-activated microglia. Here we have investigated the hitherto unknown effects of capsaicin (cap) - a transient receptor potential vanilloid 1 (TRPV1) agonist- in murine primary microglia, organotypic hippocampal slice cultures (OHSCs) and human primary monocytes. Results demonstrate that cap (0.1-25 µM) significantly (p < 0.05) inhibited the release of prostaglandin E2 (PGE2), 8-iso-PGF2α, and differentially regulated the levels of cytokines (TNF-α, IL-6 & IL-1β). Pharmacological blockade (via capsazepine & SB366791) and genetic deficiency of TRPV1 (TRPV1(-/-)) did not prevent cap-mediated suppression of PGE2 in activated microglia and OHSCs. Inhibition of PGE2 was partially dependent on the reduced levels of PGE2 synthesising enzymes, COX-2 and mPGES-1. To evaluate potential molecular targets, we discovered that cap significantly suppressed the activation of p38 MAPK and MAPKAPK2 (MK2). Altogether, we demonstrate that cap alleviates excessive inflammatory events by targeting the PGE2 pathway in in vitro and ex vivo immune cell models. These findings have broad relevance in understanding and paving new avenues for ongoing TRPV1 based drug therapies in neuroinflammatory-associated diseases.

  2. The FcepsilonRIbeta immunoreceptor tyrosine-based activation motif exerts inhibitory control on MAPK and IkappaB kinase phosphorylation and mast cell cytokine production.

    PubMed

    Furumoto, Yasuko; Nunomura, Satoshi; Terada, Tomoyoshi; Rivera, Juan; Ra, Chisei

    2004-11-19

    The high affinity IgE Fc receptor (FcepsilonRI) beta chain functions as a signal amplifier and has been linked to atopy, asthma, and allergy. Herein, we report on a previously unrecognized negative regulatory role for the nonconventional beta chain immunoreceptor tyrosine-based activation motif that contains three tyrosine residues (YX5YX3Y). Degranulation and leukotriene production was found to be impaired in cells expressing the mutated FcepsilonRIbeta immunoreceptor tyrosine-based activation motifs FYY, YYF, FYF, and FFF. In contrast, cytokine synthesis and secretion were enhanced in the YFY and FFF mutants. FcepsilonRI phosphorylation and Lyn kinase co-immunoprecipitation was intact in the YFY mutant but was lost in the FYF and FFF mutants. The phosphorylation of Syk, LAT, phospholipase gamma1/2, and Srchomology 2 domain-containing protein phosphatase 2 was intact, whereas the phosphorylation of SHIP-1 was significantly reduced in the YFY mutant cells. The FYF and FFF mutants were defective in phosphorylating all of these molecules. In contrast, the phosphorylation of ERK, p38 MAPK, IkappaB kinase beta (IKKbeta), and nuclear NFkappaB activity was enhanced in the YFY and FFF mutants. These findings show that the FcepsilonRIbeta functions to both selectively amplify (degranulation and leukotriene secretion) and dampen (lymphokine) mast cell effector responses.

  3. Protein kinase Cα suppresses Kras-mediated lung tumor formation through activation of a p38 MAPK-TGFβ signaling axis

    PubMed Central

    Hill, KS; Erdogan, E; Khoor, A; Walsh, MP; Leitges, M; Murray, NR; Fields, AP

    2014-01-01

    Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca−/− mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca−/− BASCs. In addition, LSL-Kras/Prkca−/− BASCs exhibited a modest but reproducible increase in TGFβ1 mRNA, and addition of exogenous TGFβ1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca−/− mice. Conversely, a TGFβR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca−/−BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1–3 and the Wilm’s Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFβ signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the

  4. 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

  5. Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.

    PubMed

    Massimiani, M; Vecchione, L; Piccirilli, D; Spitalieri, P; Amati, F; Salvi, S; Ferrazzani, S; Stuhlmann, H; Campagnolo, L

    2015-05-01

    Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.

  6. 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.

  7. 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.

  8. 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

  9. Inhibition of cathepsin S confers sensitivity to methyl protodioscin in oral cancer cells via activation of p38 MAPK/JNK signaling pathways

    PubMed Central

    Hsieh, Ming-Ju; Lin, Chiao-Wen; Chen, Mu-Kuan; Chien, Su-Yu; Lo, Yu-Sheng; Chuang, Yi-Ching; Hsi, Yi-Ting; Lin, Chia-Chieh; Chen, Jui-Chieh; Yang, Shun-Fa

    2017-01-01

    Oral cancer is one of the most common cancers in the world. Approximately 90% of oral cancers are subtyped to oral squamous cell carcinoma (OSCC). Despite advances in diagnostic techniques and improvement in treatment modalities, the prognosis remains poor. Therefore, an effective chemotherapy mechanism that enhances tumor sensitivity to chemotherapeutics is urgently needed. Methyl protodioscin (MP) is a furostanol bisglycoside with a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. The aim of the present study was to determine the antitumor activity of MP on OSCC and its underlying mechanisms. Our results show that treatment of OSCC cells with MP potently inhibited cell viability. Moreover, MP leading to cell cycle arrest at G2/M phase, which subsequently activates caspase-3, -8, -9 and PARP to induce cell apoptosis. Meanwhile, we also demonstrate that MP induces a robust autophagy in OSCC cells. The results indicate cathepsin S (CTSS) is involved in MP-induced apoptosis and autophagy by modulation of p38 MAPK and JNK1/2 pathways. These findings may provide rationale to combine MP with CTSS blockade for the effective treatment of OSCC. PMID:28327651

  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. Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages.

    PubMed

    Yu, Hai Yang; Kim, Kyoung-Sook; Lee, Young-Choon; Moon, Hyung-In; Lee, Jai-Heon

    2012-01-01

    Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

  12. 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.

  13. 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

  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. 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

  16. 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.

  17. 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

  18. 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

  19. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

  20. 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

  1. Profiling the MAPK/ERK dependent and independent activity regulated transcriptional programs in the murine hippocampus in vivo

    PubMed Central

    Blüthgen, Nils; van Bentum, Mirjam; Merz, Barbara; Kuhl, Dietmar; Hermey, Guido

    2017-01-01

    Activity-dependent alteration of the transcriptional program is central for shaping neuronal connectivity. Constitutively expressed transcription factors orchestrate the initial response to neuronal stimulation and serve as substrates for second messenger-regulated kinase signalling cascades. The mitogen-activated protein kinase ERK conveys signalling from the synapse to the nucleus but its genetic signature following neuronal activity has not been revealed. The goal of the present study was to identify ERK dependent and independent activity regulated transcriptional programs in the murine hippocampus. We used generalized seizures combined with the pharmacological intervention of MEK activation as an in vivo model to determine the complete transcriptional program initiated by ERK after neuronal activity. Our survey demonstrates that the induction of a large number of activity-regulated genes, including Arc/Arg3.1, Arl5b, Gadd45b, Homer1, Inhba and Zwint, is indeed dependent on ERK phosphorylation. In contrast, expression of a small group of genes, including Npas4, Arl4d, Errfi1, and Rgs2, is only partially dependent or completely independent (Ppp1r15a) of this signalling pathway. Among the identified transcripts are long non-coding (lnc) RNAs and induction of LincPint and splice variants of NEAT1 are ERK dependent. Our survey provides a comprehensive analysis of the transcriptomic response conveyed by ERK signalling in the hippocampus. PMID:28349920

  2. Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways.

    PubMed

    Santa-Cecília, Flávia V; Socias, Benjamin; Ouidja, Mohand O; Sepulveda-Diaz, Julia E; Acuña, Leonardo; Silva, Rangel L; Michel, Patrick P; Del-Bel, Elaine; Cunha, Thiago M; Raisman-Vozari, Rita

    2016-05-01

    In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson's disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1β). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.

  3. IFN-τ inhibits S. aureus-induced inflammation by suppressing the activation of NF-κB and MAPKs in RAW 264.7 cells and mice with pneumonia.

    PubMed

    Zhao, Gan; Wu, Haichong; Jiang, Kangfeng; Rui, Guangze; Zhu, Zhe; Qiu, Changwei; Guo, Mengyao; Deng, Ganzhen

    2016-06-01

    Staphylococcus aureus (S. aureus), a significant cause of pneumonia, leads to severe inflammation. Few effective treatments or drugs have been reported for S. aureus infection. Interferon tau (IFN-τ) is a type I interferon with low cellular toxicity even at high doses. Previous studies have reported that IFN-τ could significantly mitigate tissue inflammation; however, IFN-τ treatment in S. aureus-induced pneumonia has not been well reported. Thus, the aim of this study was to identify the anti-inflammatory mechanism of IFN-τ in S. aureus-induced pneumonia in mice. A S. aureus-induced pneumonia model and RAW 264.7 cells were used in this research. The histopathological as well as lung wet to dry ratio (W/D) and myeloperoxidase (MPO) activity results showed that IFN-τ could protect the lung from S. aureus damage. In addition, ELISA and qPCR revealed that IFN-τ treatment led to a decreased expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in both the cells and mouse model, but IL-10 was increased. TLR2, which is involved in the response during S. aureus infection, was also down-regulated by IFN-τ treatment and directly affected NF-κB and MAPK pathway activation. Then, we examined the phosphorylation of IκBα, NF-κB p65 and MAPKs by western blotting, and the results displayed that the phosphorylation of IκBα, NF-κB p65 and MAPKs was inhibited upon IFN-τ treatment in both the cells and mouse model. These findings indicate that IFN-τ has anti-inflammatory properties in vitro and in vivo through the inhibition of NF-κB and MAPK activation, suggesting that IFN-τ may have potential as a therapeutic agent against S. aureus-induced inflammatory diseases.

  4. 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

    Zhao, Bo-xin; Sun, Ya-bin; 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.

  5. Diverse roles for MAPK signaling in circadian clocks.

    PubMed

    Goldsmith, Charles S; Bell-Pedersen, Deborah

    2013-01-01

    The mitogen-activated protein kinase (MAPK) family of genes aids cells in sensing both extracellular and intracellular stimuli, and emerging data indicate that MAPKs have fundamental, yet diverse, roles in the circadian biological clock. In the mammalian suprachiasmatic nucleus (SCN), MAPK pathways can function as inputs allowing the endogenous clock to entrain to 24h environmental cycles. MAPKs can also interact physically and/or genetically with components of the molecular circadian oscillator, implying that MAPKs can affect the cycling of the clock. Finally, circadian rhythms in MAPK pathway activation exist in many different tissue types and in model organisms, providing a mechanism to coordinately control the expression tissue-specific target genes at the proper time of day. As such, it should probably not come as a surprise that MAPK signaling pathways and circadian clocks affect similar biological processes and defects in either pathway lead to many of the same types of human diseases, highlighting the need to better define the mechanisms that link these two fundamental pathways together.

  6. Carvacrol protects neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis by suppressing activation of MAPK/JNK-NF-κB signaling pathway

    PubMed Central

    Cui, Zhen-wen; Xie, Zheng-xing; Wang, Bao-feng; Zhong, Zhi-hong; Chen, Xiao-yan; Sun, Yu-hao; Sun, Qing-fang; Yang, Guo-yuan; Bian, Liu-guan

    2015-01-01

    Aim: Carvacrol (2-methyl-5-isopropylphenol), a phenolic monoterpene in the essential oils of the genera Origanum and Thymus, has been shown to exert a variety of therapeutic effects. Here we examined whether carvacrol protected neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis and explored the underlying mechanisms. Methods: Neuroblastoma SH-SY5Y cells were incubated with Fe2+ for 24 h, and the cell viability was assessed with CCK-8 assay. TUNEL assay and flow cytometric analysis were performed to evaluate cell apoptosis. The mRNA levels of pro-inflammatory cytokines and NF-κB p65 were determined using qPCR. The expression of relevant proteins was determined using Western blot analysis or immunofluorescence staining. Results: Treatment of SH-SY5Y cells with Fe2+ (50–200 μmol/L) dose-dependently decreased the cell viability, which was significantly attenuated by pretreatment with carvacrol (164 and 333 μmol/L). Treatment with Fe2+ increased the Bax level and caspase-3 activity, and decreased the Bcl-2 level, resulting in cell apoptosis. Furthermore, treatment with Fe2+ significantly increased the gene expression of IL-1β, IL-6 and TNF-α, and induced the nuclear translocation of NF-κB. Treatment with Fe2+ also significantly increased the phosphorylation of p38, ERK, JNK and IKK in the cells. Pretreatment with carvacrol significantly inhibited Fe2+-induced activation of NF-κB, expression of the pro-inflammatory cytokines, and cell apoptosis. Moreover, pretreatment with carvacrol inhibited Fe2+-induced phosphorylation of JNK and IKK, but not p38 and ERK in the cells. Conclusion: Carvacrol protects neuroblastoma SH-SY5Y cells against Fe2+-induced apoptosis, which may result from suppressing the MAPK/JNK-NF-κB signaling pathways. PMID:26592517

  7. Perceived health status and daily activity participation of older Malaysians.

    PubMed

    Ng, Sor Tho; Tengku-Aizan, Hamid; Tey, Nai Peng

    2011-07-01

    This article investigates the influence of perceived health status on the daily activity participation of older Malaysians. Data from the Survey on Perceptions of Needs and Problems of the Elderly, which was conducted in 1999, were used. The negative binomial regression results show that older persons with good perceived health status reported more varieties of daily activity participation, especially among the uneducated and those with below-average self-esteem. The multinomial logistic regression model suggests that older persons with good perceived health status tended to engage daily in paid work only or with leisure activities, whereas those perceived to have poor health were more likely to engage in leisure activities only or leisure and family role activities. Promotion of a healthy lifestyle at a younger age encourages every person to monitor and take responsibility for their own health, which is a necessary strategy to ensure active participation at an older age, and thus improve their well-being.

  8. Anti-metastatic activities of Antrodia camphorata against human breast cancer cells mediated through suppression of the MAPK signaling pathway.

    PubMed

    Yang, Hsin-Ling; Kuo, Yueh-Hsiung; Tsai, Ching-Tsan; Huang, Yi-Ting; Chen, Ssu-Ching; Chang, Hsueh-Wei; Lin, Elong; Lin, Wen-Hsin; Hseu, You-Cheng

    2011-01-01

    The fermented culture broth of Antrodia camphorata (A. camphorata) has been shown to promote cell cycle arrest and apoptosis of human estrogen-nonresponsive MDA-MB-231 cells. Herein, we demonstrate that non-cytotoxic concentrations (20-80 μg/mL) of A. camphorata markedly inhibited the invasion/migration of highly metastatic MDA-MB-231 cells as shown by an in vitro transwell and a wound-healing repair assay. The results of a gelatin zymography assay showed that A. camphorata suppressed the activity of matrix metalloproteinase (MMP)-9 and urokinase plasminogen activator (uPA). Western blot results demonstrated that treatment with A. camphorata decreased the expression of MMP-9, MMP-2, uPA, uPA receptor (uPAR) and vascular endothelial growth factor (VEGF); while the expression of the endogenous inhibitors of these proteins, i.e., tissue inhibitors of MMP (TIMP-1 and TIMP-2), and plasminogen activator inhibitor (PAI)-1, increased. Further investigation revealed that A. camphorata suppressed the phosphorylation of ERK1/2, p38, and JNK1/2. A. camphorata treatment also led to a dose-dependent inhibition on NF-κB binding and activation. This is the first report confirming the anti-metastatic activity of this potentially beneficial mushroom against human breast cancer.

  9. RAS/MAPK activation is associated with reduced tumor-infiltrating lymphocytes in triple-negative breast cancer: therapeutic cooperation between MEK and PD-1/PD-L1 immune checkpoint inhibitors

    PubMed Central

    Loi, Sherene; Dushyanthen, Sathana; Beavis, Paul A; Salgado, Roberto; Denkert, Carsten; Savas, Peter; Combs, Susan; Rimm, David L.; Giltnane, Jennifer M.; Estrada, Monica V.; Sánchez, Violeta; Sanders, Melinda E.; Cook, Rebecca S.; Pilkinton, Mark A.; Mallal, Simon A.; Wang, Kai; Miller, Vincent A.; Stephens, Phil J.; Yelensky, Roman; Doimi, Franco D.; Gómez, Henry; Ryzhov, Sergey V.; Darcy, Phillip K.; Arteaga, Carlos L.; Balko, Justin M.

    2015-01-01

    Purpose Tumor-infiltrating lymphocytes (TILs) in the residual disease (RD) of triple-negative breast cancers (TNBCs) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking. Experimental Design We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1-targeted immunotherapy in mouse models of breast cancer. Results Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras/MAPK signaling were significantly correlated with lower TILs. MEK inhibition up-regulated cell-surface major histocompatibility complex (MHC) expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PDL-1/PD-1 inhibition enhanced anti-tumor immune responses in mouse models of breast cancer. Conclusions These data suggest the possibility that Ras/MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1-targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors. PMID:26515496

  10. Activation of Type 4 Metabotropic Glutamate Receptor Attenuates Oxidative Stress-Induced Death of Neural Stem Cells with Inhibition of JNK and p38 MAPK Signaling

    PubMed Central

    Zhang, Zhichao; Ma, Wen; Wang, Li; Gong, Hanshi; Tian, Yumei; Zhang, Jianshui; Liu, Jianxin; Lu, Haixia

    2015-01-01

    Promoting both endogenous and exogenous neural stem cells' (NSCs) survival in the hostile host environments is essential to cell replacement therapy for central nervous system (CNS) disorders. Type 4 metabotropic glutamate receptor (mGluR4), one of the members of mGluRs, has been shown to protect neurons from acute and chronic excitotoxic insults in various brain damages. The present study investigated the preventive effects of mGluR4 on NSC injury induced by oxidative stress. Under challenge with H2O2, loss of cell viability was observed in cultured rat NSCs, and treatment with selective mGluR4 agonist VU0155041 conferred protective effects against the loss of cellular viability in a concentration-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Pretreatment of VU0155041 (30 μM) also inhibited the excessive NSC death induced by H2O2, and group III mGluRs antagonist (RS)-a-methylserine-O-phosphate (MSOP) or gene-targeted knockdown abolished the protective action of mGluR4, indicated by propidium iodide–Hoechst and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining. Western blot assay demonstrated that mGluR4 activation reversed the decreased procaspase-8/9/3and the destructed Bcl-2/Bax expressing balance, and likewise, MSOP and mGluR4 knockdown abrogated the action of mGluR4 activity. Furthermore, inhibition of JNK and p38 mitogen-activated protein kinases (MAPKs) were observed after mGluR4 activation, and as paralleling control, JNK-specific inhibitor SP600125 and p38-specific inhibitor SB203580 significantly rescued the H2O2-mediated NSC apoptosis and cleavage of procaspase-3. We suggest that activation of mGluR4 prevents oxidative stress-induced NSC death and apoptotic-associated protein activities with involvement of inhibiting the JNK and p38 pathways in cell culture. Our findings may help to develop strategies for enhancing the resided and transplanted NSC survival

  11. Emodin ameliorates lipopolysaccharide-induced mastitis in mice by inhibiting activation of NF-κB and MAPKs signal pathways.

    PubMed

    Li, Depeng; Zhang, Naisheng; Cao, Yongguo; Zhang, Wen; Su, Gaoli; Sun, Yong; Liu, Zhicheng; Li, Fengyang; Liang, Dejie; Liu, Bo; Guo, Mengyao; Fu, Yunhe; Zhang, Xichen; Yang, Zhengtao

    2013-04-05

    Emodin is an anthraquinone derivative from the Chinese herb Radix et Rhizoma Rhei. It has been reported that emodin possesses a number of biological properties, such as anti-inflammatory, anti-virus, anti-bacteria, anti-tumor, and immunosuppressive properties. However, the effect of emodin on mastitis is not yet known. The aim of this study was to investigate whether emodin has protective effect against lipopolysaccharide (LPS)-induced mastitis in a mouse model. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. Emodin was administered intraperitoneally with the dose of 1, 2, and 4 mg/kg respectively 1h before and 12h after induction of LPS. Emodin significantly reduced infiltration of neutrophilic granulocyte, activation of myeloperoxidase (MPO), concentration of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), mRNA expression levels of TNF-α, IL-1β and IL-6, which were increased in LPS-induced mouse mastitis. In addition, emodin influenced nuclear factor kappa-B signal transduction pathway by inhibiting activation of nuclear transcription factor-kappaB (NF-κB) p65 and degradation inhibitor of NF-κB α (IκBα), and emodin also influenced mitogen activated protein kinases signal transduction pathway by depression activation of p38, extracellular signal-regulated kinase (ERK), and c-jun NH2-terminal kinase (JNK). In conclusion, these results indicated that emodin could exert beneficial effects on experimental mastitis induced by LPS and may represent a novel treatment strategy for mastitis.

  12. Hair growth-promotion effects of different alternating current parameter settings are mediated by the activation of Wnt/β-catenin and MAPK pathway.

    PubMed

    Sohn, Ki Min; Jeong, Kwan Ho; Kim, Jung Eun; Park, Young Min; Kang, Hoon

    2015-12-01

    Electrical stimulation is being used in variable skin therapeutic conditions. There have been clinical studies demonstrating the positive effect of electrical stimuli on hair regrowth. However, the underlying exact mechanism and optimal parameter settings are not clarified yet. To investigate the effects of different parameter settings of electrical stimuli on hair growth by examining changes in human dermal papilla cells (hDPCs) in vitro and by observing molecular changes in animal tissue. In vitro, cultured hDPCs were electrically stimulated with different parameter settings at alternating current (AC). Cell proliferation was measured by MTT assay. The Ki67 expression was measured by immunofluorescence. Hair growth-related gene expressions were measured by RT-PCR. In animal model, different parameter settings of AC were applied to the shaved dorsal skin of rabbit for 8 weeks. Expression of hair-related genes in the skin of rabbit was examined by RT-PCR. At low voltage power (3.5 V) and low frequency (1 or 2 MHz) with AC, in vitro proliferation of hDPCs was successfully induced. A significant increase in Wnt/β-catenin, Ki67, p-ERK and p-AKT expressions was observed under the aforementioned settings. In animal model, hair regrowth was observed in the entire stimulated areas under individual conditions. Expression of hair-related genes in the skin significantly increased on the 6th week of treatment. There are optimal conditions for electrical stimulated hair growth, and they might be different in the cells, animals and human tissues. Electrical stimuli induce mechanisms such as the activation of Wnt/β-catenin and MAPK pathway in hair follicles.

  13. Alcohol promotes migration and invasion of triple-negative breast cancer cells through activation of p38 MAPK and JNK.

    PubMed

    Zhao, Ming; Howard, Erin W; Parris, Amanda B; Guo, Zhiying; Zhao, Qingxia; Yang, Xiaohe

    2017-03-01

    Although alcohol is an established breast cancer risk factor, the underlying mechanisms remain unclear. Previous studies examined the general association between alcohol consumption and breast cancer risk; however, the risk for different breast cancer subtypes has been rarely reported. Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking hormone receptors and HER2 expression, and having poor prognosis. Understanding the molecular mechanisms of TNBC etiology remains a significant challenge. In this study, we investigated cellular responses to alcohol in two TNBC cell lines, MDA-MB-231 and MDA-MB-468. Our results showed that alcohol at low concentrations (0.025-0.1% v/v) induced cell proliferation, migration, and invasion in 1% FBS-containing medium. Molecular analysis indicated that these phenotypic changes were associated with alcohol-induced reactive oxygen species production and increased p38 and JNK phosphorylation. Likewise, p38 or JNK inhibition attenuated alcohol-induced cell migration and invasion. We revealed that alcohol treatment activated/phosphorylated NF-κB regulators and increased transcription of NF-κB-targeted genes. While examining the role of acetaldehyde, the major alcohol metabolite, in alcohol-associated responses in TNBC cells, we saw that acetaldehyde induced cell migration, invasion, and increased phospho-p38, phospho-JNK, and phospho-IκBα in a pattern similar to alcohol treatment. Taken together, we established that alcohol promotes TNBC cell proliferation, migration, and invasion in vitro. The underlying mechanisms involve the induction of oxidative stress and the activation of NF-κB signaling. In particular, the activation of p38 and JNK plays a pivotal role in alcohol-induced cellular responses. These results will advance our understanding of alcohol-mediated development and promotion of TNBC. © 2016 Wiley Periodicals, Inc.

  14. 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

  15. Peptidoglycan-mediated IL-8 expression in human alveolar type II epithelial cells requires lipid raft formation and MAPK activation.

    PubMed

    Cheon, In Su; Woo, Sang Su; Kang, Seok-Seong; Im, Jintaek; Yun, Cheol-Heui; Chung, Dae Kyun; Park, Dong Ki; Han, Seung Hyun

    2008-03-01

    Staphylococcus aureus, a major sepsis-causing Gram-positive bacterium, invades pulmonary epithelial cells and causes lung diseases. In the lung, alveolar type II epithelial cells play an important role in innate immunity by secreting chemokines and antimicrobial peptides upon bacterial infection whereas type I cells mainly function in gas-exchange. In this study, we investigated the ability of S. aureus peptidoglycan (PGN) to induce expression of a chemokine, IL-8, in a human alveolar type II epithelial cell line, A549. PGN induces IL-8 mRNA and protein expression in a dose- and time-dependent manner. Supplementation of soluble CD14 further enhanced the PGN-induced IL-8 expression. Interestingly, PGN-induced IL-8 expression was inhibited by nystatin, a specific inhibitor for lipid rafts, but not by chlorpromazine, a specific inhibitor for clathrin-coated pits. Furthermore, PGN-induced IL-8 expression was attenuated by inhibitors for MAP kinases such as ERK, p38 kinase, and JNK/SAPK, whereas no inhibitory effect was observed by inhibitors for reactive oxygen species or protein kinase C. Electrophoretic mobility shift assay demonstrates that PGN increased the DNA binding of the transcription factors, AP-1 and NF-kappaB while minimally, NF-IL6, all of which are involved in the transcription of IL-8. Taken together, these results suggest that PGN induces IL-8 expression in a CD14-enhanced manner in human alveolar type II epithelial cells, through the formation of lipid rafts and the activation of MAP kinases, which ultimately leads to activation of AP-1, NF-kappaB, and NF-IL6.

  16. Glutamate excitotoxicity activates the MAPK/ERK signaling pathway and induces the survival of rat hippocampal neurons in vivo.

    PubMed

    Ortuño-Sahagún, Daniel; González, Raúl Montes; Verdaguer, Ester; Huerta, Verónica Chaparro; Torres-Mendoza, Blanca M; Lemus, Lourdes; Rivera-Cervantes, Martha Catalina; Camins, A; Zárate, C Beas

    2014-03-01

    Current knowledge concerning the molecular mechanisms of the cellular response to excitotoxic insults in neurodegenerative diseases is insufficient. Although glutamate (Glu) has been widely studied as the main excitatory neurotransmitter and principal excitotoxic agent, the neuroprotective response enacted by neurons is not yet completely understood. Some of the molecular participants have been revealed, but the signaling pathways involved in this protective response are just beginning to be identified. Here, we demonstrate in vivo that, in response to the cell damage and death induced by Glu excitotoxicity, neurons orchestrate a survival response through the extracellular signal-regulated kinase (ERK) signaling pathway by increasing ERK expression in the rat hippocampal (CA1) region, allowing increased neuronal survival. In addition, this protective response is specifically reversed by U0126, an ERK inhibitor, which promotes cell death only when it is administered together with Glu. Our findings demonstrate that the ERK signaling pathway has a neuroprotective role in the response to Glu-induced excitotoxicity in hippocampal neurons. Therefore, the ERK signaling pathway may be activated as a cellular response to excitotoxic injury to prevent damage and neural loss, representing a novel therapeutic target in the treatment of neurodegenerative diseases.

  17. Pheophorbide a-mediated photodynamic therapy induces autophagy and apoptosis via the activation of MAPKs in human skin cancer cells.

    PubMed

    Yoon, Hyo-Eun; Oh, Seone-Hee; Kim, Soo-A; Yoon, Jung-Hoon; Ahn, Sang-Gun

    2014-01-01

    Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitizer that can induce significant antitumor effects in several types of tumor cells. The present study investigated the mechanism of Pa-mediated photodynamic therapy (Pa-PDT) in the human skin cancer cell lines A431 and G361. PDT significantly inhibited the cell growth in a Pa-concentration-dependent manner. We observed increased expression of Beclin-1, LC3B and ATG5, which are markers of autophagy, after PDT treatment in A431 cells but not in G361 cells. In G361 cells, Pa-PDT strongly induced PARP cleavage and subsequent apoptosis, which was confirmed using Annexin V/Propidium iodide double staining. Pa-PDT predominantly exhibited its antitumor effects via activation of ERK1/2 and p38 in A431 and G361 cells, respectively. An in vivo study using the CAM xenograft model demonstrated that Pa-PDT strongly induced autophagy and apoptosis in A431-transplanted tumors and/or apoptosis in G361-transplanted tumors. These results may provide a basis for understanding the underlying mechanisms of Pa-PDT and for developing Pa-PDT as a therapy for skin cancer.

  18. MAPK1/3 regulate hepatic lipid metabolism via ATG7-dependent autophagy.

    PubMed

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

    2016-01-01

    Although many biological functions of MAPK1/ERK2-MAPK3/ERK1 (mitogen-activated protein kinase 1/3) have been reported, a direct effect of MAPK1/3 on hepatic lipid metabolism remains largely unknown. We recently showed that activation of MAPK1/3 ameliorates liver steatosis in LEPR (leptin receptor)-deficient (db/db) mice, a classic animal model for liver steatosis. Consistent with these results, knockdown of MAPK1/3 promotes liver steatosis in C57/B6J wild-type (WT) mice. Autophagic flux and ATG7 (autophagy related 7) levels are increased by MAPK1/3 activation or decreased by MAPK1/3 knockdown in livers and primary hepatocytes. Blockade of autophagic flux by chloroquine (CQ) or ATG7 knockdown reverses the ameliorated liver steatosis in MAPK1/3-activated db/db mice. Together, these findings identify a beneficial role for MAPK1/3 in liver steatosis that is mediated by ATG7-dependent autophagy, which provides novel insights into the mechanisms underlying liver steatosis and create a rationale for targeting MAPK1/3 in the treatment of liver steatosis.

  19. 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.

  20. The present status of stellar activity theory

    NASA Astrophysics Data System (ADS)

    Belvedere, G.

    Features, predictions available from, and problems facing mean field electrodynamic (MFE) models of solar and stellar dynamo activities are outlined. The linear, kinetic dynamo (LKD) approach describes the interaction between a turbulent velocity field and a magnetic field in terms of an MFE induction equation. LKD permits the reciprocal generation of poloidal and toroidal fields through rotational and cyclonic, turbulent forces. Nonlinear hydromagnetic theory considers magnetic field back reaction effects on the kinematic field by the Lorentz EM body force. All forces in the LKD model increase in impact with advancing spectral evolution. Differential rotation and dynamo action become particularly important. Further research is needed to account for the gap in the F-G region of the evolutionary continuum, the occurrence of multimodal dynamo features, and the presence of cyclic and noncyclic dynamo activity, depending on the star.

  1. 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

  2. Pseudoginsenoside-F11 (PF11) exerts anti-neuroinflammatory effects on LPS-activated microglial cells by inhibiting TLR4-mediated TAK1/IKK/NF-κB, MAPKs and Akt signaling pathways.

    PubMed

    Wang, Xiaoxiao; Wang, Chunming; Wang, Jiming; Zhao, Siqi; Zhang, Kuo; Wang, Jingmin; Zhang, Wei; Wu, Chunfu; Yang, Jingyu

    2014-04-01

    Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been shown to possess significant neuroprotective activity. Since microglia-mediated inflammation is critical for induction of neurodegeneration, this study was designed to investigate the effect of PF11 on activated microglia. PF11 significantly suppressed the release of ROS and proinflammatory mediators induced by LPS in a microglial cell line N9 including NO, PGE2, IL-1β, IL-6 and TNF-α. Moreover, PF11 inhibited interaction and expression of TLR4 and MyD88 in LPS-activated N9 cells, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. PF11 also inhibited the phosphorylation of Akt and MAPKs induced by LPS in N9 cells. Importantly, PF11 significantly alleviated the death of SH-SY5Y neuroblastoma cells and primary cortical neurons induced by the conditioned-medium from activated microglia. At last, the effect of PF11 on neuroinflammation was confirmed in vivo: PF11 mitigated the microglial activation and proinflammatory factors expression obviously in both cortex and hippocampus in mice injected intrahippocampally with LPS. These findings indicate that PF11 exerts anti-neuroinflammatory effects on LPS-activated microglial cells by inhibiting TLR4-mediated TAK1/IKK/NF-κB, MAPKs and Akt signaling pathways, suggesting its therapeutic implication for neurodegenerative disease associated with neuroinflammation.

  3. Activin suppresses LPS-induced Toll-like receptor, cytokine and inducible nitric oxide synthase expression in normal human melanocytes by inhibiting NF-κB and MAPK pathway activation.

    PubMed

    Kim, Young Il; Park, Seung-Won; Kang, In Jung; Shin, Min Kyung; Lee, Mu-Hyoung

    2015-10-01

    Activins are dimeric growth and differentiation factors that belong to the transforming growth factor (TGF)-β superfamily of structurally related signaling proteins. In the present study, we examined the mechanisms through which activin regulates the lipopolysaccharide (LPS)-induced transcription of Toll-like receptors (TLRs), cytokines and inducible nitric oxide synthase (iNOS) in human melanocytes, as well as the involvement of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling. Cell proliferation was analyzed by cell viability assay, mRNA expression was detected by RT-qPCR, and protein expression was measured by western blot analysis. LPS increased the mRNA expression of TLRs (TLR1-10) and cytokines [interleukin (IL)-1β, IL-6, IL-8 and TNF-α], as well as the mRNA and protein expression of iNOS. Activin decreased the LPS-induced TLR and cytokine mRNA expression, as well as the LPS-induced iNOS mRNA and protein expression. In addition, activin suppressed NF-κB p65 activation and blocked inhibitor of NF-κB (IκBα) degradation in LPS-stimulated melanocytes, and reduced LPS-induced p38 MAPK and MEK/ERK activation. On the whole, our results demonstrated that activin inhibited TLR and cytokine expression in LPS-activated normal human melanocytes and suppressed LPS-induced iNOS gene expression. Moreover, the anti-inflammatory effects of activin were shown to be mediated through the suppression of NF-κB and MAPK signaling, resulting in reduced TLR and iNOS expression, and in the inhibition of inflammatory cytokine expression.

  4. Field Operations Program Activities Status Report

    SciTech Connect

    J. E. Francfort; D. V. O'Hara; L. A. Slezak

    1999-05-01

    The Field Operations Program is an electric vehicle testing and evaluation program sponsored by US Department of Energy and managed by the Idaho National Engineering and Environmental Laboratory. The Program's goals are to evaluate electric vehicles in real-world applications and environments, support electric vehicle technology advancement, develop infrastructure elements necessary to support significant electric vehicle use, support increased use of electric vehicles in federal fleets, and increase overall awareness and acceptance of electric vehicles. This report covers Program activities from fiscal year 1997 through mid-fiscal year 1999. The Field Operations Program succeeded the Site Operator Program, which ended in September 1996. Electric vehicle testing conducted by the Program includes baseline performance testing (EV America testing), accelerated reliability (life-cycle) testing, and fleet testing. The baseline performance parameters include accelerations, braking, range, energy efficiency, and charging time. The Program collects accelerated reliability and fleet operations data on electric vehicles operated by the Program's Qualified Vehicle Testing (QVT) partners. The Program's QVT partners have over 3 million miles of electric vehicle operating experience.

  5. 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.

  6. Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells.

    PubMed

    de Bittencourt Pasquali, Matheus Augusto; Gelain, Daniel Pens; Zeidán-Chuliá, Fares; Pires, André Simões; Gasparotto, Juciano; Terra, Silvia Resende; Moreira, José Cláudio Fonseca

    2013-04-01

    As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 μM) or therapeutic (5, 10 or 20 μM). Retinol at 10 and 20 μM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 μM), SB203580 (10 μM) or siRNA to either p38α (MAPK14) or p38β (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 μg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by

  7. VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System

    PubMed Central

    Bankapalli, Leela K.; Mishra, Rahul C.; Raychaudhuri, Saumya

    2017-01-01

    VopE, a mitochondrial targeting T3SS effector protein of Vibrio cholerae, perturbs innate immunity by modulating mitochondrial dynamics. In the current study, ectopic expression of VopE was found to be toxic in a yeast model system and toxicity was further aggravated in the presence of various stressors. Interestingly, a VopE variant lacking predicted mitochondrial targeting sequence (MTS) also exhibited partial lethality in the yeast system. With the aid of yeast genetic tools and different stressors, we have demonstrated that VopE and its derivative VopEΔMTS modulate cell wall integrity (CWI-MAPK) signaling pathway and have identified several critical residues contributing to the lethality of VopE. Furthermore, co-expression of two effectors VopEΔMTS and VopX, interfering with the CWI-MAPK cellular pathway can partially suppress the VopX mediated yeast growth inhibition. Taken together, these results suggest that VopE alters signaling through the CWI-MAPK pathway, and demonstrates the usefulness of yeast model system to gain additional insights on the functionality of VopE. PMID:28373966

  8. Influence of dominance status on adrenal activity and ovarian cyclicity status in captive African elephants.

    PubMed

    Proctor, Christine M; Freeman, Elizabeth W; Brown, Janine L

    2010-01-01

    The North American African (Loxodonta africana) elephant population is not self-sustaining, in part because of a high rate of abnormal ovarian activity. About 12% of adult females exhibit irregular cycles and 31% do not cycle at all. Our earlier work revealed a relationship between dominance status and ovarian acyclicity, with dominant females being more likely to not cycle normally. One theory is that dominant females may be expending more energy to maintaining peace within the captive herd than for supporting reproduction. The goal of this study was to determine if there was a relationship among dominance status, serum cortisol concentrations, and ovarian acyclicity. We hypothesized that adrenal glucocorticoid activity would be increased in dominant, noncycling elephants as compared with subdominant individuals. Blood samples were collected weekly over a 2-year period in 81 females of known dominance and cyclicity status, and analyzed for cortisol. Based on a path analysis model (Reticular Action Model Or Near Approximation [RAMONA]), noncycling, dominant African elephant females did not have higher mean serum cortisol concentrations, or exhibit more variability (i.e., coefficient of variation, standard deviation) in cortisol secretion. This study suggests that alterations in adrenal activity are not related to dominance status nor contribute directly to acyclicity in captive African elephants.

  9. 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

  10. Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway.

    PubMed

    Park, Ga Bin; Kim, Yeong Seok; Lee, Hyun-Kyung; Song, Hyunkeun; Cho, Dae-Ho; Lee, Wang Jae; Hur, Dae Young

    2010-12-15

    CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

  11. Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system.

    PubMed

    dos-Santos, Guilherme Rodrigo Reis Monteiro; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida; de Oliveira, Pedro Lagerblad; Nepomuceno-Silva, José Luciano; de Melo, Luiz Dione Barbosa; Araujo, Helena Maria Marcolla; Lopes, Ulisses Gazos

    2015-11-06

    The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

  12. Whole transcriptome profiling of the human hippocampus suggests an involvement of the KIBRA rs17070145 polymorphism in differential activation of the MAPK signaling pathway.

    PubMed

    Piras, I S; Krate, J; Schrauwen, I; Corneveaux, J J; Serrano, G E; Sue, L; Beach, T G; Huentelman, M J

    2017-04-05

    The rs17070145-T variant of the WWC1 gene, coding for the KIBRA protein, has been associated with both increased episodic memory performance and lowered risk for late onset Alzheimer's disease, although the mechanism behind this protective effect has not been completely elucidated. To achieve a better understanding of the pathways modulated by rs17070145 and its associated functional variant(s), we used laser capture microdissection (LCM) and RNA-sequencing to investigate the effect of rs17070145 genotypes on whole transcriptome expression in the human hippocampus (HP) of 22 neuropathologically normal individuals, with a specific focus on the dentate gyrus (DG) and at the pyramidal cells (PC) of CA1 and CA3 sub-regions. Differential expression analysis of RNA-seq data within the HP based on the rs17070145 genotype revealed an overexpression of genes involved in the MAPK signaling pathway, potentially driven by the T/T genotype. The most important contribution comes from genes dysregulated within the DG region. Other genes significantly dysregulated, and not involved in the MAPK1 pathway (Adj P < 0.01 and Fold Change > |1.00|) were: RSPO4 (HP); ARC, DUSP5, DNAJB5, EGR4, PPP1R15A, WBP11P1, EGR1, GADD45B (DG); CH25H, HSPA1A, HSPA1B, TNFSF9 and NPAS4 (PC). Several evidences suggested that the MAPK signaling pathway is linked with memory and learning processes. In non-neuronal cells, the KIBRA protein is phosphorylated by ERK1/2 (involved in the MAPK signaling) in cells as well as in vitro. Several of the other dysregulated genes are involved in memory and learning processes, as well as in Alzheimer's Disease. In conclusion, our results suggest that the effect of the WWC1 rs17070145 polymorphism on memory performance and Alzheimer's disease might be due to a differential regulation of the MAPK signaling, a key pathway involved in memory and learning processes. This article is protected by copyright. All rights reserved.

  13. Bullied Status and Physical Activity in Texas Adolescents

    ERIC Educational Resources Information Center

    Case, Kathleen R.; Pérez, Adriana; Saxton, Debra L.; Hoelscher, Deanna M.; Springer, Andrew E.

    2016-01-01

    This study examined the association between having been bullied at school during the past 6 months ("bullied status") and not meeting physical activity (PA) recommendations of 60 minutes of daily PA during the past week among 8th- and 11th-grade Texas adolescents. Multiple logistic regression analysis was conducted to examine this…

  14. Esculin exhibited anti-inflammatory activities in vivo and regulated TNF-α and IL-6 production in LPS-stimulated mouse peritoneal macrophages in vitro through MAPK pathway.

    PubMed

    Niu, Xiaofeng; Wang, Yu; Li, Weifeng; Zhang, Hailin; Wang, Xiumei; Mu, Qingli; He, Zehong; Yao, Huan

    2015-12-01

    Esculin, a coumarinic derivative found in Aesculus hippocastanum L. (Horse-chestnut), has been reported to have potent anti-inflammatory properties. The present study is designed to investigate the protective effects of esculin on various inflammation models in vivo and in vitro and to clarify the possible mechanism. Induced-animal models of inflammation and lipopolysaccharide (LPS)-challenged mouse peritoneal macrophages were used to examine the anti-inflammatory activity of esculin. In present study, xylene-induced mouse ear edema, carrageenan-induced rat paw edema, and carrageenan-induced mouse pleurisy were attenuated by esculin. In vitro, the pro-inflammatory cytokine levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatant were reduced by esculin. Meanwhile, we found that esculin significantly inhibited LPS-induced activation of mitogen-activated protein kinase (MAPK) pathway in peritoneal macrophages. These results suggest that esculin has potent anti-inflammatory activities in vivo and in vitro, which may involve the inhibition of the MAPK pathway. Esculin may be a promising preventive agent for inflammatory diseases in human.

  15. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  16. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  17. PsMPK7, a stress-associated mitogen-activated protein kinase (MAPK) in Phytophthora sojae, is required for stress tolerance, reactive oxygenated species detoxification, cyst germination, sexual reproduction and infection of soybean.

    PubMed

    Gao, Jian; Cao, Mingna; Ye, Wenwu; Li, Haiyang; Kong, Liang; Zheng, Xiaobo; Wang, Yuanchao

    2015-01-01

    The sensing of stress signals and their transduction into appropriate responses are crucial for the adaptation, survival and infection of phytopathogenic fungi and oomycetes. Amongst evolutionarily conserved pathways, mitogen-activated protein kinase (MAPK) cascades function as key signal transducers that use phosphorylation to convey information. In this study, we identified a gene, designated PsMPK7, one of 14 predicted genes encoding MAPKs in Phytophthora sojae. PsMPK7 was highly transcribed in each tested stage, but was up-regulated in the zoospore, cyst and cyst germination stages. Silencing of PsMPK7 affected the growth of germinated cysts, oospore production and the pathogenicity of soybean. PsMPK7 transcription was induced by stresses from sorbitol, NaCl and hydrogen peroxide. Transformants in which PsMPK7 expression was silenced (PsMPK7-silenced) were significantly more sensitive to osmotic and oxidative stress. Aniline blue and diaminobenzidine staining revealed that the silenced lines did not suppress the host reactive oxygen species (ROS) burst, indicating that either the inoculated plants activated stronger defence responses to the transformants and/or the PsMPK7-silenced transformants failed to overcome plant defences. In addition, extracellular secretion of laccase decreased in the silenced lines. Overall, our results indicate that the PsMPK7 gene encodes a stress-associated MAPK in P. sojae that is important not only for responses to various stresses, but also for ROS detoxification, cyst germination, sexual oospore production and infection of soybean.

  18. IGF-I/PI3K/Akt and IGF-I/MAPK/ERK pathways in vivo in skeletal muscle are regulated by nutrition and contribute to somatic growth in the fine flounder.

    PubMed

    Fuentes, Eduardo N; Björnsson, Björn Thrandur; Valdés, Juan Antonio; Einarsdottir, Ingibjörg Eir; Lorca, Belen; Alvarez, Marco; Molina, Alfredo

    2011-06-01

    The insulin-like growth factor-I (IGF-I) is a key regulator of skeletal muscle growth in vertebrates, promoting mitogenic and anabolic effects through the activation of the MAPK/ERK and the PI3K/Akt signaling pathways. Nutrition also affects skeletal muscle growth, activating intracellular pathways and inducing protein synthesis and accretion. Thus, both hormonal and nutritional signaling regulate muscle mass. In this context, plasma IGF-I levels and the activation of both pathways in response to food were evaluated in the fine flounder using fasting and refeeding trials. The present study describes for the first time in a nonmammalian species that the MAPK/ERK and PI3K/Akt are activated by exogenous circulating IGF-I, as well as showing that the MAPK/ERK pathway activation is modulated by the nutritional status. Also, these results show that there is a time-dependent regulation of IGF-I plasma levels and its signaling pathways in muscle. Together, these results suggest that the nutritionally managed IGF-I could be regulating the activation of the MAPK/ERK and the PI3K/Akt signaling pathways differentially according to the nutritional status, triggering different effects in growth parameters and therefore contributing to somatic growth in fish. This study contributes to the understanding of the nutrient regulation of IGF-I and its signaling pathways in skeletal muscle growth in nonmammalian species, therefore providing insight concerning the events controlling somatic growth in vertebrates.

  19. Physical activity and physical activity adherence in the elderly based on smoking status.

    PubMed

    Cooper, Theodore V; Resor, Michelle R; Stoever, Colby J; Dubbert, Patricia M

    2007-10-01

    This study assessed the impact of current smoking status and lifetime smoking status on physical fitness and physical activity regimen adherence as part of a larger study on walking for exercise in elderly primary care patients at a Veterans Affairs Medical Center. At baseline, 218 participants self-reported smoking status which was verified by carbon monoxide expiration. Former and current smokers responded to questions about length of time quit, average daily cigarette intake, and years a smoker. Smoking measures were re-collected at 6- and 12-month follow-ups if the participants indicated a change in smoking status. Veterans completed multiple measures of physical activity (e.g., 6-min walk, 7-day Physical Activity Recall), and adherence to a physical activity goal was assessed. The Physical Component Summary (PCS) subscale of the Medical Outcomes Study Short Form-36 (MOS SF-36) was used to assess health-related quality of life. Hierarchical regression models indicated smoking status was a predictor of the baseline 6-min walk such that smokers walked significantly shorter distances than nonsmokers. In addition, smoking status was found to be a significant predictor of adherence; however, the overall model that included smoking status as a predictor did not demonstrate a significant effect on adherence. Neither smoking status nor pack years were predictors of baseline self-reported physical activity or changes in physical activity post intervention. Results are consistent with recommendations to use physical exercise as an aid to tobacco cessation, even in aging men with extensive smoking histories.

  20. Antiallergic Activity of Ethanol Extracts of Arctium lappa L. Undried Roots and Its Active Compound, Oleamide, in Regulating FcεRI-Mediated and MAPK Signaling in RBL-2H3 Cells.

    PubMed

    Yang, Woong-Suk; Lee, Sung Ryul; Jeong, Yong Joon; Park, Dae Won; Cho, Young Mi; Joo, Hae Mi; Kim, Inhye; Seu, Young-Bae; Sohn, Eun-Hwa; Kang, Se Chan

    2016-05-11

    The antiallergic potential of Arctium lappa L. was investigated in Sprague-Dawley rats, ICR mice, and RBL-2H3 cells. Ethanol extract (90%) of A. lappa (ALE, 100 μg/mL) inhibited the degranulation rate by 52.9%, determined by the level of β-hexosaminidase. ALE suppressed passive cutaneous anaphylaxis (PCA) in rats and attenuated anaphylaxis and histamine release in mice. To identify the active compound of ALE, we subsequently fractionated and determined the level of β-hexosaminidase in all subfractions. Oleamide was identified as an active compound of ALE, which attenuated the secretion of histamine and the production of tumor necrosis factor (TNF)-α and interleukin-4 (IL-4) in cells treated with compound 48/80 or A23187/phorbol myristate acetate (PMA). Oleamide suppressed FcεRI-tyrosine kinase Lyn-mediated pathway, c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinases (p38-MAPKs). These results showed that ALE and oleamide attenuated allergic reactions and should serve as a platform to search for compounds with antiallergic activity.

  1. LeMAPK1, LeMAPK2, and LeMAPK3 are associated with nitric oxide-induced defense response against Botrytis cinerea in the Lycopersicon esculentum fruit.

    PubMed

    Zheng, Yanyan; Hong, Hui; Chen, Lin; Li, Jingyuan; Sheng, Jiping; Shen, Lin

    2014-02-12

    Nitric oxide (NO) and mitogen-activated protein kinases (MAPKs) are signal molecules involved in the disease resistance of plants. To investigate the role of tomato MAPKs in the NO-mediated defense response, mature green tomatoes (Lycopersicon esculentum Mill. cv. Qian-xi) were treated with a MAPKs inhibitor (1,4-diamino-2,3-dicyano-1,4-bis(o-amino-phenylmercapto) butadiene (U0126)), NO donor sodium nitroprusside (SNP), and SNP plus U0126. Treatment with U0126 increased the incidence of disease and size of lesion areas in the tomato fruits after being inoculated with Botrytis cinerea. NO enhanced the resistance of the tomato fruits against Botrytis cinerea invasion and the activities of nitric oxide synthase, Chitinase, β-1,3-glucanase, polyphenol oxidase, and phenylalanine ammonia-lyase. However, the effects of NO on disease resistance were weakened by the MAPKs inhibitor. Meanwhile, the relative expression of LeMAPK1, LeMAPK2, and LeMAPK3 in the (SNP + U0126)-treated fruits was lower than that in the SNP-treated fruits. The results suggest that LeMAPK1/2/3 are involved in NO-induced disease resistance of tomato fruits against Botrytis cinerea.

  2. Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity.

    PubMed

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-12-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches.

  3. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-{kappa}B translocation

    SciTech Connect

    Jawan, Bruno; Kao, Y.-H.; Goto, Shigeru; Pan, M.-C.; Lin, Y.-C.; Hsu, L.-W.; Nakano, Toshiaki; Lai, C.-Y.; Sun, C.-K.; Cheng, Y.-F.; Tai, M.-H.

    2008-06-15

    Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 {mu}M after 48 h incubation. Pretreatment with 100 {mu}M PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and I{kappa}B{alpha}, as well as the nuclear translocation of NF-{kappa}B primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-{kappa}B nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.

  4. Reduced signaling of PI3K-Akt and RAS-MAPK pathways are the key targets for weight loss-induced cancer prevention by dietary calorie restriction and/or physical activity

    PubMed Central

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-01-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics, and lipidomics were employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into 4 groups for 10 weeks: ad lib-fed sedentary control, ad lib-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE), and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics, and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67, and 110 genes were significantly changed in DCR, PE, and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including upregulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears the reduction of both Ras-MAPK and PI3K-Akt signaling pathways are cancer preventive targets that have been consistently demonstrated by three bioinformatics approaches. PMID:25283328

  5. ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration.

    PubMed

    Huo, Y-N; Chen, W; Zheng, X-X

    2015-11-08

    Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

  6. Expression profile of mitrogen-activated protein kinase (MAPK) signaling genes in the skeletal muscle & liver of rat with type 2 diabetes: Role in disease pathology

    PubMed Central

    Tang, Xiaoli; Deng, Libin; Xiong, Huangui; Li, Guilin; Lin, Jiari; Liu, Shuangmei; Xie, Jinyan; Liu, Jun; Kong, Fanjun; Tu, Guihua; Peng, Haiying; Liang, Shangdong

    2014-01-01

    Background & objectives: Type 2 diabetes (T2D) is characterized as hyperglycaemia caused by defects in insulin secretion, and it affects target tissues, such as skeletal muscle, liver and adipose tissue. Therefore, analyzing the changes of gene expression profiles in these tissues is important to elucidate the pathogenesis of T2D. We, therefore, measured the gene transcript alterations in liver and skeletal muscle of rat with induced T2D, to detect differentially expressed genes in liver and skeletal muscle and perform gene-annotation enrichment analysis. Methods: In the present study, skeletal muscle and liver tissue from 10 streptozotocin-induced diabetic rats and 10 control rats were analyzed using gene expression microarrays. KEGG pathways enriched by differentially expressed genes (DEGs) were identified by WebGestalt Expander and GATHER software. DEGs were validated by the method of real-time PCR and western blot. Results: From the 9,929 expressed genes across the genome, 1,305 and 997 differentially expressed genes (DEGs, P<0.01) were identified in comparisons of skeletal muscle and liver, respectively. Large numbers of DEGs (200) were common in both comparisons, which was clearly more than the predicted number (131 genes, P<0.001). For further interpretation of the gene expression data, three over-representation analysis softwares (WebGestalt, Expander and GATHER) were used. All the tools detected one KEGG pathway (MAPK signaling) and two GO (gene ontology) biological processes (response to stress and cell death), with enrichment of DEGs in both tissues. In addition, PPI (protein-protein interaction) networks constructed using human homologues not only revealed the tendency of DEGs to form a highly connected module, but also suggested a “hub” role of p38-MAPK-related genes (such as MAPK14) in the pathogenesis of T2D. Interpretation & conclusions: Our results indicated the considerably aberrant MAPK signaling in both insulin-sensitive tissues of T2D rat

  7. TGF{beta}1 induces apoptosis in invasive prostate cancer and bladder cancer cells via Akt-independent, p38 MAPK and JNK/SAPK-mediated activation of caspases

    SciTech Connect

    Al-Azayzih, Ahmad; Gao, Fei; Goc, Anna; Somanath, Payaningal R.

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer TGF{beta} induced apoptosis in invasive prostate cancer and bladder cancer cells. Black-Right-Pointing-Pointer TGF{beta} inhibited prostate/bladder cancer cell proliferation and colony/foci formation. Black-Right-Pointing-Pointer TGF{beta} induced prostate/bladder cancer cell apoptosis independent of Akt inhibition. Black-Right-Pointing-Pointer TGF{beta} inhibited ERK1/2 phosphorylation in prostate/bladder cancer cells. Black-Right-Pointing-Pointer TGF{beta} induced p38 MAPK and JNK-mediated activation of caspases-9, -8 and -3. -- Abstract: Recent findings indicate that advanced stage cancers shun the tumor suppressive actions of TGF{beta} and inexplicably utilize the cytokine as a tumor promoter. We investigated the effect of TGF{beta}1 on the survival and proliferation of invasive prostate (PC3) and bladder (T24) cancer cells. Our study indicated that TGF{beta}1 decreased cell viability and induced apoptosis in invasive human PC3 and T24 cells via activation of p38 MAPK-JNK-Caspase9/8/3 pathway. Surprisingly, no change in the phosphorylation of pro-survival Akt kinase was observed. We postulate that TGF{beta}1 pathway may be utilized for specifically targeting urological cancers without inflicting side effects on normal tissues.

  8. Extracellular serglycin upregulates the CD44 receptor in an autocrine manner to maintain self-renewal in nasopharyngeal carcinoma cells by reciprocally activating the MAPK/β-catenin axis

    PubMed Central

    Chu, Qiaoqiao; Huang, Hongbing; Huang, Tiejun; Cao, Li; Peng, Lixia; Shi, Simei; Zheng, Lisheng; Xu, Liang; Zhang, Shijun; Huang, Jialing; Li, Xinjian; Qian, Chaonan; Huang, Bijun

    2016-01-01

    Serglycin is a proteoglycan that was first found to be secreted by hematopoietic cells. As an extracellular matrix (ECM) component, serglycin promotes nasopharyngeal carcinoma (NPC) metastasis and serves as an independent, unfavorable NPC prognostic indicator. The detailed mechanism underlying the roles of serglycin in cancer progression remains to be clarified. Here, we report that serglycin knockdown in NPC cells inhibited cell sphere formation and tumor seeding abilities. Serglycin downregulation enhanced high-metastasis NPC cell sensitivity to chemotherapy. It has been reported that serglycin is a novel ligand for the stem cell marker CD44. Interestingly, we found a positive correlation between serglycin expression and CD44 in nasopharyngeal tissues and NPC cell lines. Further study revealed that CD44 was an ERK-dependent downstream effector of serglycin signaling, and serglycin activated the MAPK/β-catenin axis to induce CD44 receptor expression in a positive feedback loop. Taken together, our novel findings suggest that ECM serglycin upregulated CD44 receptor expression to maintain NPC stemness by interacting with CD44 and activating the MAPK/β-catenin pathway, resulting in NPC cell chemoresistance. These findings suggest that the intervention of serglycin/CD44 axis and downstream signaling pathway is a rational strategy for targeting NPC cancer stem cell therapy. PMID:27809309

  9. Urea and ureolytic activity in lakes of different trophic status.

    PubMed

    Siuda, Waldemar; Chróst, Ryszard J

    2006-01-01

    Urea and uraease (U-ase) activity were determined in water samples taken from the surface layers of 17 lakes of different trophic status. Urea concentrations were inversely correlated with the trophic status<